Construct capable of release in closed circular form from a larger nucleotide sequence permitting site specific expression and/or developmentally regulated expression of selected genetic sequences

ABSTRACT

The present invention relates generally to constructs and in particular genetic constructs comprising polynucleotide sequences capable of release in covalently closed, circular form from a larger nucleotide sequence such as, but not limited to, a genome of a eukaryotic cell. Preferably, once released, a polynucleotide sequence is reconstituted in a form which permits expression of the polynucleotide sequence. In one embodiment, the reconstituted polynucleotide sequence comprises a coding sequence with all or part of an extraneous nucleotide such as, but not limited to, an intronic sequence or other splice signal inserted therein. Expression and in particular transcription of the coding sequence involves splicing out the extraneous sequence. The release and circularization is generally in response to a stimulus such as a protein-mediated stimulus. More particularly, the protein is a viral or prokaryotic or eukaryotic derived protein or developmentally and/or tissue specific regulated protein.

FIELD OF THE INVENTION

The present invention relates generally to constructs and in particulargenetic constructs comprising polynucleotide sequences capable ofrelease in covalently closed, circular form from a larger nucleotidesequence such as, but not limited to, a genome of a eukaryotic cell.Preferably, once released, a polynucleotide sequence is reconstituted ina form which permits expression of the polynucleotide sequence. In oneembodiment, the reconstituted polynucleotide sequence comprises a codingsequence with all or part of an extraneous nucleotide such as, but notlimited to, intronic sequence or other splice signal inserted therein.Expression and in particular transcription of the coding sequenceinvolves splicing out the extraneous sequence. According to thisembodiment, the coding sequence may encode a peptide, polypeptide orprotein, an antisense or sense nucleic acid molecule or a ribozyme. Inanother embodiment, the reconstituted polynucleotide sequence willcomprise the extraneous polynucleotide sequence located between apromoter element and coding sequence. In this embodiment, expression ofthe coding sequence is not substantially adversely affected by thepresence of the extraneous sequence. In still a further embodiment, thereconstituted sequence forms an RNA promoter or other regulatory geneticsequence comprising the extraneous sequence which does not affect thefunction of the promoter. The release and circularization is generallyin response to a stimulus such as a protein-mediated stimulus. Moreparticularly, the protein is a viral or prokaryotic or eukaryoticderived protein or developmentally and/or tissue specific regulatedprotein. The construct of the present invention is particularly usefulin conferring genetic resistance against pathogens or inducing apoptosisor other cell death mechanisms useful, for example, in treating canceror inducing male or female sterility in plants. The constructs permit,therefore, site specific expression and/or developmentally regulatedexpression of selected genetic sequences.

BACKGROUND OF THE INVENTION

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any othercountry.

The increasing sophistication of recombinant DNA technology is greatlyfacilitating research and development in a range of technological fieldssuch as the medical, horticultural and agricultural industries. Ofparticular importance, is the exploitation of naturally occurringgenetic mechanisms in, for example, pathogens, to induce usefulphenotypic changes in cells, such as plant cells. This is particularlyevident in the horticultural field in relation to mechanisms to induceresistance in plants to a range of plant pathogens.

There has been considerable progress in the development of virusresistance in crops, for example, through transformation with transgenesderived from the target viruses. The most successful use of transgeneshas been with RNA plant viruses. However, despite a number attempts tocontrol single stranded DNA (ssDNA) plant viruses through transgenicresistance, strategies which have been successful for RNA plant viruseshave not been as effective for ssDNA plant viruses. DNA plant virusesand particularly the single stranded DNA (ssDNA) plant viruses areresponsible for significant commercial losses in a wide range of fruit,vegetable, grain and fibre crops in the tropics and sub-tropics.

There have been two groups of ssDNA viruses which infect plants. Theseare the Geminiviridae and the recently described nanovirus group.Members of the Geminiviridae have geminate virions and either amonopartite or bipartite circular ssDNA genome. Each molecule is about2.7 kb in length. Of the Geminiviridae genera, the begomoviruses and themastreviruses are the most important. The begomoviruses arewhitefly-transmitted and have either monopartite or bipartite genomes.Members of this genus include some of the most economically devastatingviruses of modem agriculture such as tomato (yellow) leaf curl(consisting of a range of different viruses spread through most tropicaland sub-tropical regions), African cassaya mosaic (Africa), bean goldenmosaic (South and Central America), mungbean yellow mosaic (India) andcotton leaf curl (South and South-East Asia) viruses. The impact of manyof the begomoviruses has increased dramatically over recent years as aresult of the widespread introduction of the aggressive “B biotype” ofthe whitefly vector, Bemesia tabaci. The mastreviruses have had a lesserimpact on agriculture but are responsible for significant losses in somecrops. These viruses are transmitted by the leafhoppers and havemonopartite genomes. Members of this genus include maize streak(Africa), wheat dwarf (Europe) and tobacco yellow dwarf (Australia)viruses.

The nanoviruses have isometric virions and circular ssDNA genomes butthese genomes are multi-component with at least six different integralgenomic components each of which is approximately 1 kb. These virusesare transmitted by aphids except for one tentative nanovirus, coconutfoliar decay virus, which is transmitted by a treehopper and has onlybeen reported from Vanuatu. The economically most important nanovirus isbanana bunchy top virus (BBTV) which nearly destroyed the Australianbanana industry in the 1920s and causes major losses in the SouthPacific, Asia and Africa. Subterranean clover stunt (Australia), fababean necrotic yellows (Mediterranean) and coconut foliar decay (Vanuatu)viruses all cause significant yield loss.

The genome organization of the begomoviruses, the mastreviruses and thenanoviruses have significant differences including the number and sizeof genomic components and number and size of genes, the processing oftranscripts, the orientation of genes and the like. There are, however,remarkable similarities which suggest that these viruses have verysimilar replication and infection strategies. All the gemini- andnanoviruses encode (i) a Rep protein which has nicking and joiningactivity and directs rolling circle replication of the viral genome;(ii) a virion coat protein; (iii) a protein that is involved in bindinghost cell retinoblastoma-like proteins resulting in the cell moving to Sphase; (iv) a cell-to-cell movement protein; and (v) a nuclear shuttleprotein. Further, the viruses have functionally similar intergenicregions (IR). For instance, the IR of begomovimses, the LIR ofmastreviruses and the CR-SL of banana bunchy top nanovirus all contain(1) a stem/loop structure, the nonanucleotide loop sequence of which ishighly conserved between all gemini- and nanoviruses and is the site ofnicking and ligation by the Rep protein; and (ii) a domain within thisregion which recognizes the Rep protein. The SIR of the mastrevirusesand the CR-M of banana bunchy top nanovirus are the site of binding ofan endogenous primer responsible for priming the conversion of virionssDNA into transcriptionally active dsDNA.

The success in developing transgenic resistance to RNA viruses in cropsand the increasing demand for such resistance to ssDNA viruses hasresulted in investigation of a wide range of strategies for ssDNAviruses targeting various viral genes including the coat protein gene,movement protein gene and the Rep protein gene. In addition, strategiesusing defective interfering DNAs and a suicide gene have beeninvestigated. Most work in this area has involved begomoviruses ratherthan mastre- or nanoviruses.

In work leading up to the present invention, the inventors haveexploited the replication mechanisms of ssDNA viruses in order to inducegenetic resistance in plants. However, the present invention has wideranging applications in modulating genetic activities such as expressionof polynucleotide sequences to effect a particular phenotype in responseto a stimulus.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1, <400>2, etc. A sequence listing isprovided after the claims.

One aspect of the present invention provides a construct comprising agenetic element operably flanked by nucleotide sequences recognizable bya viral-derived, replication-facilitating protein or its derivatives oreukaryotic and prokaryotic cell homologues when integrated into thegenome of a eukaryotic cell which viral-derived,replication-facilitating protein or its derivatives or eukaryotic orprokaryotic cell homologues facilitates excision and circularization ofthe genetic element and all or part of the flanking nucleotide sequencesand wherein said nucleotide sequences recognizable by saidviral-derived, replication-facilitating protein or its derivatives oreukaryotic or prokaryotic cell homologues are adjacent to or insertedwithin one or more extraneous sequences including intron sequences orparts thereof or other splice signals wherein the genetic element andother nucleotide sequences, in a non-circular form, comprise two modularnucleotide sequences which, upon circularization, form a geneticsequence exhibiting a property or a capacity for exhibiting a propertyabsent in the two modular nucleotide sequences prior to circularizationor prior to circularization and expression.

Another aspect of the present invention provides a construct comprisinga genetic element flanked by Rep-protein recognition sequences orfunctional homologues from other viruses or eukaryotic or prokaryoticcells which facilitate the generation of a circular nucleotide sequencecomprising said genetic element in the presence of a Rep protein or itsfunctional derivatives or homologues wherein said Rep-proteinrecognition sequences are adjacent to or inserted within one or morerecognition sequences, said genetic element comprising a polynucleotidesequence operably linked to regulatory sequences required to permitexpression of said polynucleotide sequence when said genetic element iscontained within a circularized molecule wherein the genetic element inlinear form comprises in the 5′ to 3′ order:—

a polynucleotide sequence; and

regulatory sequences to permit expression of said polynucleotidesequence when in circular form,

such that upon circularization the genetic element comprises theregulatory sequence separated from the polynucleotide sequence by all orpart of a Rep protein recognition sequence wherein upon expression, saidpolynucleotide sequence encodes an expression product.

A further aspect of the present invention provides a constructcomprising in 5′ to 3′ order first, second, third, fourth, fifth andsixth nucleotide sequences wherein:

the first and sixth nucleotide sequences may be the same or differentand each comprises a Rep protein-recognition sequence capable of beingrecognized by one or more Rep proteins or derivatives or homologuesthereof such that genetic material flanked by said first and sixthsequences including all or part of said first and sixth sequences whensaid construct is integrated in a larger nucleotide sequence such as agenomic sequence, is capable of being excised and circularized whereinsaid Rep-protein recognition sequences are adjacent to or insertedwithin one or more extraneous sequences including intronic sequences orparts thereof or other splice signals;

the second nucleotide sequence comprises a 3′ portion of polynucleotidesequence;

the third nucleotide sequence is a transcription terminator orfunctional derivative or homologue thereof operably linked to saidsecond sequence;

the fourth nucleotide sequence is a promoter sequence operably linked tothe fifth nucleotide sequence; and

the fifth nucleotide sequence is a 5′ portion of a polynucleotidesequence wherein the 5′ and 3′ portions of said polynucleotide sequencerepresent a full coding sequence of said polynucleotide sequence;

wherein in the presence of one or more Rep proteins, when the constructis integrated into a larger nucleotide sequence such as a genomicsequence, a circularized genetic sequence is generated separate fromsaid larger nucleotide sequence comprising in order said promotersequence operably linked to a polynucleotide sequence comprising all orpart of the extraneous sequence or other splice signal comprising all orpart of said first and/or sixth nucleotide sequences and a transcriptionterminator sequence.

Still another aspect of the present invention is directed to a geneticelement for use in generating a construct, said genetic elementcomprising in 5′ to 3′ direction, a 3′ portion of a polynucleotidesequence operably linked to a transcription terminator; a promoteroperably linked to a 5′ portion of a polynucleotide sequence whereinupon circularization, the 5′ portion of the polynucleotide sequence isoperably linked to said 3′ portion of the polynucleotide sequenceseparated by all or part of an extraneous sequence or intron sequence orother splice signal.

Yet another aspect of the present invention provides a constructcomprising the nucleotide sequence substantially as set forth in SEQ IDNO:31 to SEQ ID NO:36 or a nucleotide sequence having 60% similarity toeach of SEQ ID NO:31 to SEQ ID NO:36 or a nucleotide sequence capable ofhybridizing to one or more of SEQ ID NO:31 to SEQ ID NO:36 or acomplementary form thereof under low stringency conditions at 42° C.

Even yet another aspect of the present invention contemplates a methodfor generating a transgenic plant or progeny thereof resistant to assDNA virus, said method comprising introducing into the genome of saidplant a construct comprising in the 5′ to 3′ order, a Repprotein-recognition sequence adjacent to or within an intronic sequenceor other splice signal, a 3′ end portion of a polynucleotide sequence, atranscription terminator or its functional equivalent, a promotersequence operably linked to a 5′ end portion of the polynucleotidesequence wherein the 5′ and 3′ portions of the polynucleotide sequencerepresent the coding region of a peptide, polypeptide or protein capableof inducing cell death or dormancy, and same or different Repprotein-recognition sequences; wherein upon infection of said plantcells by ssDNA virus having a Rep protein which is capable ofrecognizing the flanking Rep protein-recognition sequences, theconstruct is excised and circularizes thus reconstituting saidpolynucleotide sequence in a form which is expressed into a peptide,polypeptide or protein which kills the plant cell or otherwise rendersthe plant cell dormant.

Even still another aspect of the present invention provides a constructcomprising a genetic element flanked by a Rep protein-recognitionsequences which facilitate the generation of a circular nucleotidesequence comprising said genetic element in the presence of a Repprotein or its functional derivatives or homologues wherein saidRep-protein recognition sequences are adjacent to or inserted within oneor more extraneous sequences including intronic sequences or partsthereof or other splice signal, said genetic element comprising a 3′portion and a 5′ portion of a promoter separated by a length of anucleotide sequence to substantially prevent functioning of saidpromoter, said genetic element in linear form comprises in the 5′ to 3′order:—

a 3′ portion of said promoter;

optionally a polynucleotide sequence operably linked to said 3′ portionof said promoter; and

a 5′ portion of said promoter,

such that upon circularization the genetic element comprises the 5′ and3′ portions of the promoter sequence separated by all or part of a Repprotein-recognition sequence and/or intron sequences or other splicesignal but which does not inactivate the activity of the promoter, saidcircular molecule optionally further comprising the promoter operablylinked to polynucleotide sequence.

The promoter may be a DNA promoter or an RNA promoter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation of pTBN.

FIG. 2 is a diagrammatic representation of pTBN6.

FIG. 3 is a diagrammatic representation of pTBN1.

FIG. 4 is a diagrammatic representation of pRTBN6.

FIG. 5 is a diagrammatic representation of pRTBN1.

FIG. 6 is a diagrammatic representation of pRTBN 1/6.

FIG. 7 is a schematic representation of plasmid p35S-2301.

FIG. 8 is a schematic representation of plasmid pTEST1.

FIG. 9 is a schematic representation of plasmid pTEST2.

FIG. 10 is a schematic representation of plasmid pTEST3.

FIG. 11 is schematic representation of plasmid pTEST4.

FIG. 12 is a schematic representation of plasmid pBI-TYDV1.1mer.

FIG. 13 is a schematic representation of plasmid p35S-Rep.

FIG. 14 is a graphical representation of the results of a cell deathassay using expression vectors. Error bars show 95% confidenceintervals.

FIG. 15 is a graphical representation of the results ofrecircularization cell death assay using expression vectors. Error barsshow 95% confidence intervals.

FIG. 16 is a graphical representation of the results from the induciblerecircularization cell death assay using expression vectors. Error barsshow 95% confidence intervals.

FIG. 17 is a schematic representation of plasmids (A) p35S-BTR-LIR and(B) p35S-BUTR-LIR.

FIG. 18 is a schematic representation of plasmids (A) p35S-BTR and (B)p35S-BUTR.

FIG. 19 is a schematic representation of plasmids (A) pBTR.test1 and (B)pBUTRtest1.

FIG. 20 is a diagrammatic representation of pGI.

FIG. 21 is a diagrammatic representation of pGI6.

FIG. 22 is a diagrammatic representation of pGI1.

FIG. 23 is a diagrammatic representation of pRGI6.

FIG. 24 is a diagrammatic representation of pRGI1.

FIG. 25 is a diagrammatic representation of pRGI 1/6.

FIG. 26 is a schematic representation of a proposed model forRep-activated expression of human serum albumin from plasmid pHSA1.

FIG. 27 is a diagrammatic representation of a construct for use insense/antisense modulation of genetic expression.

A summary of sequence identifiers is provided herewith.

SUMMARY OF SEQUENCE IDENTIFIERS

SEQUENCE IDENTIFIER DESCRIPTION SEQ ID NO: 1 to SEQ ID NO: 18 Syntheticoligonucleotide SEQ ID NO: 19 to SEQ ID NO: 44 Primers SEQ ID NO: 45 toSEQ ID NO: 52 Synthetic oligonucleotide SEQ ID NO: 66 Barnase pTBN SEQID NO: 67 Barnase pRTBN6 SEQ ID NO: 68 Barnase pRTBN1 SEQ ID NO: 69 GFPpGI SEQ ID NO: 70 GFP pGI6 SEQ ID NO: 71 GFP pGI1 SEQ ID NO: 72 primerSEQ ID NO: 73 primer

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the recognition that aviral-derived, replication-facilitating protein may be used to exciseand circularize specific, targeted sequences from the genome of aeukaryotic cell. The term “excise” includes in this case release andmore particularly replicative release of targeted sequences. Theviral-derived, replication-facilitating proteins initiate nicking,excision and circularization of genetic elements flanked by particularsequences specific for and recognized by the viral-derived,replication-facilitating protein. The present invention extends toderivatives of the viral-derived facilitating proteins and eukaryoticand prokaryotic homologues thereof. The present invention extends to,therefore, any sequences capable of facilitating cleavage and ligationof polynucleotide sequences and are referred to hereinafter as“recognition sequences” and may also be considered herein as “extraneoussequences”. The recognition sequences are adjacent to or inserted withinextraneous sequences including intronic sequences or other splicesignals.

In one embodiment, the circularization process permits the formation ofa particular genetic sequence from two modular components separated by asplicable extraneous sequence upon expression. Prior to circularization,the modular components are genetically separated and hence not operablylinked. Operable linkage is conveniently shown, for example, in oneembodiment, by the ability for the genetic sequence comprising themodular components to be expressed. The term “expressed” in thisinstance includes transcription to an mRNA sequence and optionallytranslation of the mRNA sequence to a translation product. Expression,however, is not the sole criterion for constitution of a geneticsequence from modular components. In another embodiment, the geneticsequence produced following circularization may have other usefulfunctions not requiring expression. For example, the resulting geneticsequence may comprise a protein binding recognition sequence therebytargeting particular cytoplasmic or nuclear proteins. Alternatively, thereconstituted polynucleotide sequence comprises an extraneous sequencebetween the promotors elements and a coding sequence. In the case of theformer, the promoter is preferably an RNA promoter such as from a TMV,AMV or TEV virus. Alternatively, the promoter is a DNA promoter wherethe insertion of the recognition does not substantially adversely affectits activity.

Accordingly, one aspect of the present invention provides a constructcomprising a genetic element operably flanked by nucleotide sequencesrecognizable by a viral-derived, replication-facilitating protein or itsderivatives or eukaryotic and prokaryotic cell homologues whenintegrated into the genome of a eukaryotic cell which viral-derived,replication-facilitating protein or its derivatives or eukaryotic orprokaryotic cell homologues facilitates excision and circularization ofthe genetic element and all or part of the flanking nucleotide sequencesand wherein said nucleotide sequences recognizable by saidviral-derived, replication-facilitating protein or its derivatives oreukaryotic or prokaryotic cell homologues are adjacent to or insertedwithin one or more extraneous sequences including intron sequences orparts thereof or other splice signals wherein the genetic element andother nucleotide sequences, in a non-circular form, comprise two modularnucleotide sequences which, upon circularization, form a geneticsequence exhibiting a property or a capacity for exhibiting a propertyabsent in the two modular nucleotide sequences prior to circularizationor prior to circularization and expression.

The term “construct” is used in its broadest sense and includes agenetic construct, nucleic acid molecule, vector, plasmid or any othernucleotide sequence comprising at least two heterologous sequences. Theconstruct, therefore, is a recombinant molecule engineered to comprisetwo or more nucleotide sequences from different genetic sources. In oneembodiment, the construct is in an isolated form. The term “isolated”includes biologically pure, substantially pure or in another conditionwhere at least one purification step has been performed on a samplecomprising the construct. A “purification step” includes, for example, aprecipitation, centrifugation and/or a chromatographic orelectrophoretic separation. In another embodiment, the genetic constructis integrated into the genome of a host cell. The construct may comprisenucleotide sequences which are lost, removed or rearranged followingintegration. In yet another embodiment, the construct is in circularform either generated in vitro or following excision from the genome ofthe host cell.

The term “genetic element” is used in its broadest sense and includes aseries of two or more nucleotide sequences engineered in a particularorder relative to the 5′ to 3′ or 3′ to 5′ orientations of the geneticelement. In essence, the genetic element comprises two nucleotidesequences in modular form. The term “modular” is not to impart anylimitation to the construction or structure of the nucleotide sequencesbut emphasizes that a single genetic sequence is divided into twocomponents, i.e. modular components. Upon circularization, the twomodular components are orientated together to constitute, after removalof any extraneous sequences including intronic and splice sequences orother recognition sequences, a single genetic sequence exhibiting aparticular activity or property not present when the genetic sequence isin separate modular form. In certain circumstances, extraneous sequencesintervening the two modular components when in circular form may notneed to be removed if their presence does not substantially adverselyaffect the function of the modular components when constituted in thecorrect orientation relative to each other after circularization.Generally, the modular components are referred to as 5′ portions and 3′portions of a polynucleotide sequence. A portion comprises from a fewnucleotides (i.e. from about 2 to about 500) to many (i.e. from about501 to about 10,000). The 5′ and 3′ portions may encompass a centralportion.

In a preferred embodiment, the genetic element comprises, when incircular form, a promoter operably linked to the genetic sequencecomprising the two modular sequences. The two modular sequences may beseparated by an intronic sequence, splice signal or other recognitionsequence. The genetic element comprises, therefore, in linear form inthe 5′ to 3′ direction, a first modular nucleotide sequence comprisingthe 3′ portion of a polynucleotide sequence, a promoter sequenceoperably linked to the 5′ portion of the above-mentioned polynucleotidesequence. Upon circularization, the 3′ portion of the polynucleotidesequence is now orientated and fused by base linkage to the 5′ portionof the polynucleotide sequence thus reconstituting a functionalpolynucleotide sequence operably linked to a promoter. Depending on theconstruct, an intronic sequence, splice signal or other recognitionsequence may separate the 5′ and 3′ portions of the reconstitutedpolynucleotide sequence. Upon processing during expression, the intronicsequence, splice signal or other recognition sequence may be excised. Ina particularly preferred embodiment, the genetic element comprises atranscription terminator sequence operably linked and downstream of the3′ portion of the polynucleotide sequence. Terms such as “promoter” and“terminator” are used in their broadest sense and are described in moredetail below.

In another embodiment, the reconstituted polynucleotide sequence encodesan intronic, splice signal or other recognition sequence located betweenthe promoter element and the coding sequence. According to thisembodiment, the recognition sequence would not be removed duringtranscription.

In yet another alternative embodiment, the genetic element comprises twomodular components of a promoter or other regulatory sequence.Preferably, the modular components form a promoter sequence aftercircularization. If an intronic sequence, splice signal or otherrecognition sequence separates the modular components of a promotersequence, then such a sequence does not destroy or partially destroy theactivity of the promoter sequence. Alternatively, the promoter is an RNApromoter such as a promoter from TMV, AMV or TEV.

The polynucleotide sequence, when reconstituted, exhibits an activity orproperty or a capacity to exhibit an activity or property not present inthe separate modular nucleotide sequences prior to fusion followingcircularization. Such an activity or property includes the ability toencode a peptide, polypeptide or protein having a particular function,the ability to encode a mRNA sequence which may subsequently betranslated into a peptide, polypeptide or protein or which may act as anantisense or sense molecule for down-regulation of a host gene or othergenetic sequence or acting as a promoter or other regulatory sequence.Another property contemplated by the genetic sequences includes theability to bind to protein to interact with nucleic regulatory sequencesor to act or encode ribozyme and/or deoxyribozyme molecules.

Of particular importance, the genetic sequence may encode proteinshaving enzymic activity, regulatory activity or structural activity orexhibit a therapeutic activity if administered to a mammal such as ahuman or livestock animal. Examples of the latter type of moleculeinclude cytokines, interferons and growth factors. Proteins havingenzymic activity are particularly preferred and such proteins are usefulin activating a biochemical pathway, facilitating the flow ofmetabolites down a particular pathway, conferring a property such asresistance to an insecticide, fungicide or herbicide or conferringresistance to a pathogen such as an intracellular pathogen includingviruses and intracellular microorganisms. Cells contemplated as targetsfor the genetic construct of the present invention include animal cells,plant cells, unicellular organisms and microorganisms. Animal cells maybe from primates, hmans, livestock animals, avian speices, fish,reptiles, amphibians and insects and arachnids. Plant cells may be frommonocotyledonas or dicotyledonas plants.

In one particularly useful embodiment, the peptide, polypeptide orprotein encoded by the polynucleotide sequence induces apoptosis orother form of cell death. This is particularly useful as a means offacilitating genetic resistance to viruses, for example, or formediating cell death of particular types of cells.

In one embodiment, for example, the construct is used to facilitateresistance to a single stranded DNA (ssDNA) virus. Such viruses causeconsiderable damage to the agricultural and horticultural industries byinfecting important crop and ornamental plants. Two groups of ssDNAviruses which infect plants are the gemini- and nanoviruses.

In one embodiment, the flanking sequences recognizable by aviral-derived, replication-facilitating protein or its derivatives orprokaryotic or eukaryotic cell homologues are stem/loop nucleotidestructures. Preferably, the stem/loop structures comprise a shortnucleotide sequence loop of from about 5 to about 20, preferably fromabout 6 to about 15 and most preferably about 9 nucleotides (i.e.nonanucleotide) and which is the site of nicking and ligation by theviral-derived, replication-facilitating protein or its derivatives orprokaryotic or eukaryotic cell homologues. In another embodiment, theflanking sequences are recognized by any protein having cleavage andligation activity. An example of such a protein is topoisomerase. Allthese sequences are referred to herein as “recognition sequences”. Mostpreferably, the recognition sequences are recognized by the “Rep”protein. This protein is derived from members of the geminiviridae andnanoviruses and binds to a 5′ domain on a stem/loop structure comprisingthe recognition sequence. The present invention, however, is not limitedto the use of a stem loop structure although such use is contemplatedherein. The present invention extends to any Rep protein from ageminivirus or nanovirus as well as derivatives thereof or homologuesfrom other viruses or from eukaryotic or prokaryotic cells. Examples ofeukaryotic cells include mammalian, insect, reptilian; amphibian andyeast cells.

Examples of other recognition sequences or their equivalents include theintergenic regions of BBTV DNA 1-6, the short and long repeats of TLCVor TYDV. An “intronic sequence” is a sequence of nucleotides which,following transcription, have the capacity to be spliced out. In certaincircumstances, the intronic sequence is not spliced out such as when thepresence of the intronic sequence does not adversely affect thefunctioning of the sequence into which the intronic sequence isinserted.

The construct of this aspect of the present invention may comprise thesame or substantially the same recognition sequences as flankingsequences, that is, the sequences recognizable by a single Rep proteinor its derivatives or homologues or may comprise different recognitionsequences recognizable by different Rep proteins or derivatives thereofor eukaryotic or prokaryotic cell homologues thereof. Furthermore, therecognition sequences may be full sequences or part sequences such astwo half intronic sequences.

The Rep protein may be introduced to a cell such as following viralinfection or be encoded by genetic sequences developmentally or tissuespecifically expressed in the animal or plant or organism which carriesthe construct.

In another preferred embodiment, there is provided a constructcomprising a genetic element flanked by Rep-protein recognitionsequences or functional homologues from other viruses or eukaryotic orprokaryotic cells which facilitate the generation of a circularnucleotide sequence comprising said genetic element in the presence of aRep protein or its functional derivatives or homologues wherein saidRep-protein recognition sequences are adjacent to or inserted within oneor more recognition sequences, said genetic element comprising apolynucleotide sequence operably linked to regulatory sequences requiredto permit expression of said polynucleotide sequence when said geneticelement is contained within a circularized molecule wherein the geneticelement in linear form comprises in the 5′ to 3′ order:—

-   -   a polynucleotide sequence; and    -   regulatory sequences to permit expression of said polynucleotide        sequence when in circular form,        such that upon circularization the genetic element comprises the        regulatory sequence separated from the polynucleotide sequence        by all or part of a Rep protein-recognition sequence wherein        upon expression, said polynucleotide sequence encodes an        expression product.

Preferably, the regulatory sequences include or comprise a promotersequence and optionally a transcription terminator. As stated above, arecognition sequence includes an extraneous sequence such as an intronicsequence or other splice signal.

In another embodiment, there is provided a construct comprising agenetic element flanked by a Rep protein-recognition sequences whichfacilitate the generation of a circular nucleotide sequence comprisingsaid genetic element in the presence of a Rep protein or its functionalderivatives or homologues wherein said Rep-protein recognition sequencesare adjacent to or inserted within one or more extraneous sequencesincluding intronic sequences or parts thereof or other splice signals,said genetic element comprising a 3′ portion and a 5′ portion of apromoter separated by a length of a nucleotide sequence to substantiallyprevent functioning of said promoter, said genetic element in linearform comprises in the 5′ to 3′ order:—

-   -   a 3′ portion of said promoter;    -   optionally a polynucleotide sequence operably linked to said 3′        portion of said promoter; and    -   a 5′ portion of said promoter, such that upon circularization        the genetic element comprises the 5′ and 3′ portions of the        promoter sequence separated by all or part of a Rep        protein-recognition sequence but which does not inactivate the        activity of the promoter, said circular molecule optionally        further comprising the promoter operably linked to        polynucleotide sequence.

Alternatively, the promoter is an RNA promoter such as from TMV, TEV orAMV.

An advantage of such a system is that when the construct is in linearform and, for example, integrated into a larger nucleotide sequence suchas a genome, the promoter sequence is inactive. However, uponcircularization, the promoter sequence is reconstituted thus permittingpromoter activity. The optionally present operably linked polynucleotidesequence is then expressed.

Examples of suitable promoters include the cauliflower mosaic virus 35Spromoter. Another useful promoters is the ubiquitin promoter. Generally,monocot promoters such as the ubiquitin promoter require an intronicsequence between the promoter and the start codon of the expressed exon.Absent this intronic sequence, expression of the promoter is either verylow or completely lacking. The genetic construct of the presentinvention may be designed such that upon circularization, the intronicsequence comprising the stem loop structure forms an intronic sequencedownstream of the ubiquitin promoter thus permitting its operation.

Other suitable promoters are described below.

In another preferred embodiment, the present invention provides aconstruct comprising in 5′ to 3′ order first, second, third, fourth,fifth and sixth nucleotide sequences wherein:

the first and sixth nucleotide sequences may be the same or differentand each comprises a Rep protein-recognition sequence capable of beingrecognized by one or more Rep proteins or derivatives or homologuesthereof such that genetic material flanked by said first and sixthsequences including all or part of said first and sixth sequences whensaid construct is integrated in a larger nucleotide sequence such as agenomic sequence, is capable of being excised and circularized whereinsaid Rep-protein recognition sequences are adjacent to or insertedwithin one or more extraneous sequences including intronic sequences orparts thereof or other splice signals;

the second nucleotide sequence comprises a 3′ portion of polynucleotidesequence;

the third nucleotide sequence is a transcription terminator orfunctional derivative or homologue thereof operably linked to saidsecond sequence;

the fourth nucleotide sequence is a promoter sequence operably linked tothe fifth nucleotide sequence; and

the fifth nucleotide sequence is a 5′ portion of a polynucleotidesequence wherein the 5′ and 3′ portions of said polynucleotide sequencerepresent a full coding sequence of said polynucleotide sequence;

wherein in the presence of one or more Rep proteins, when the constructis integrated into a larger nucleotide sequence such as a genomicsequence, a circularized genetic sequence is generated separate fromsaid larger nucleotide sequence comprising in order said promotersequence operably linked to a polynucleotide sequence comprising all orpart of the extraneous sequence or other splice signal comprising all orpart of said first and/or sixth nucleotide sequences and a transcriptionterminator sequence.

In accordance with the above-mentioned aspect of the present invention,the first and sixth nucleotide sequences represent recognition sequencesfor a viral-derived, replication-facilitating protein such as Rep orderivatives thereof or eukaroytic or prokaryotic derivatives thereofadjacent to or inserted within an intronic sequence or other splicesignal. The second to fifth nucleotide sequences represent the geneticelements previously defined.

Yet another aspect of the present invention is directed to a geneticelement for use in generating a construct, said genetic elementcomprising in 5′ to 3′ direction, a 3′ portion of a polynucleotidesequence operably linked to a transcription terminator; a promoteroperably linked to a 5′ portion of a polynucleotide sequence whereinupon circularization, the 5′ portion of the polynucleotide sequence isoperably linked to said 3′ portion of the polynucleotide sequenceseparated by all or part of an extraneous sequence or intron sequence orother splice signal.

The constructs of the present invention have a range of applications. Inone embodiment, the construct is used to generate genetic resistance inplant cells to ssDNA viruses. The particular viruses for whichprotection is sought against include but not limited to geminivirus ornanovirus. In this embodiment, the construct comprises a “suicide gene”,i.e. a gene encoding a product which induces cell apoptosis, lysis,death or a state of biochemical or physiological dormancy. The constructis introduced into a plant cell under conditions to permit integrationinto the plant cell genome. A plant is regenerated from the plant celland propagated when the plant is infected by a particular ssDNA virushaving a Rep protein which recognizes the Rep protein-recognitionsequences flanking the genetic element of the construct, the constructis excised and recircularizes thus reconstituting the “suicide gene” andfacilitating its expression. The cell then dies or otherwise becomesdormant thus preventing the replication and release of ssDNA viruses.

In a particularly preferred embodiment, the present invention provides aconstruct comprising the nucleotide sequence substantially as set forthin SEQ ID NO:31 to SEQ ID NO:36 or a nucleotide sequence having 60%similarity to each of SEQ ID NO:31 to SEQ ID NO:36 or a nucleotidesequence capable of hybridizing to one or more of SEQ ID NO:31 to SEQ IDNO:36 or a complementary form thereof under low stringency conditions at42° C.

Accordingly, another aspect of the present invention contemplates amethod for generating a transgenic plant or progeny thereof resistant toa ssDNA virus, said method comprising introducing into the genome ofsaid plant a construct comprising in the 5′ to 3′ order, a Repprotein-recognition sequence adjacent to or within an intronic sequenceor other splice signal, a 3′ end portion of a polynucleotide sequence, atranscription terminator or its functional equivalent, a promotersequence operably linked to a 5′ end portion of the polynucleotidesequence wherein the 5′ and 3′ portions of the polynucleotide sequencerepresent the coding region of a peptide, polypeptide or protein capableof inducing cell death or dormancy, and same or different Repprotein-recognition sequences; wherein upon infection of said plantcells by ssDNA virus having a Rep protein which is capable ofrecognizing the flanking Rep protein-recognition sequences, theconstruct is excised and circularizes thus reconstituting saidpolynucleotide sequence in a form which is expressed into a peptide,polypeptide or protein which kills the plant cell or otherwise rendersthe plant cell dormant.

Another use of the instant construct is to produce male sterile plants.In this embodiment, a gene encoding a Rep protein is placed under thecontrol of a pollen-specific promoter. A construct comprising the abovedescribed “suicide gene” is also generated using Rep protein-recognitionsequences recognized by the Rep gene under the control of thepollen-specific promoter. When pollen is formed, the pollen-specificpromoter is activated thus activating the suicide gene. Pollen cells arethen selectively destroyed or rendered dormant.

Other uses of the construct herein described include introducing geneticmaterial facilitating a colour change into plants or specific tissue orseeds or other reproductive material of plants. An example of a geneticsequence facilitating a colour change is a gene encoding an enzyme of aanthocyanin pathway such as a flavonal 3′-hydroxylase, flavonal3′,5′-hydroxylase, or flavone 3′-synthase.

In another embodiment, the construct may be flanked by two different Repprotein-recognition sequences, i.e. recognized by two different Repproteins. One Rep protein may then be encoded by a gene inserted intothe plant genome and the other Rep protein may be introduced by aninfecting ssDNA virus. Alternatively, the Rep proteins may be encoded bydifferent promoters which are expressed and certain development stages.

Although the present invention is particularly described in relation toplants and ssDNA viruses, the present invention extends to homologousexcision structures and other proteins with site-specific excision andjoining activities from other sources such as non-ssDNA viruses andeukaryotic cells such as insect, mammalian or reptilian cells. Insofaras the present invention relates to plants, the plants may bemonocotyledonous or dicotyledonous plants.

The term “plant cell” as used herein includes protoplasts or other cellsderived from plants, gamete-producing cells and cells which regenerateinto whole plants. Plant cells include cells in plants as well asprotoplasts or other cells in culture.

The term “polynucleotide” or “nucleic acid” as used herein designatesmRNA, RNA, cRNA, cDNA or DNA.

“Polypeptide”, “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues and to variants of same.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence identity”, “percentage of sequenceidentity” and “substantial identity”. A “reference sequence” is at least12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that the two polynucleotides,sequence comparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment of atleast 6 contiguous positions, usually about 50 to about 100, moreusually about 100 to about 150 in which a sequence is compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. The comparison window may compriseadditions or deletions (i.e., gaps) of about 20% or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Optimal alignment ofsequences for aligning a comparison window may be conducted bycomputerised implementations of algorithms (GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Drive Madison, Wis., USA) or by inspectionand the best alignment (i.e., resulting in the highest percentagehomology over the comparison window) generated by any of the variousmethods selected. Reference also may be made to the BLAST family ofprograms as for example disclosed by Altschul et al., 1997. A detaileddiscussion of sequence analysis can be found in Unit 19.3 of Ausubel etal., 1994-1998.

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. For the purposes of the present invention, “sequence identity”will be understood to mean the “match percentage” calculated by theDNASIS computer program (Version 2.5 for windows; available from HitachiSoftware engineering Co., Ltd., South San Francisco, Calif., USA) usingstandard defaults as used in the reference manual accompanying thesoftware.

Reference herein to a low stringency includes and encompasses from atleast about 0 to at least about 15% v/v formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Generally,low stringency is at from about 25-30° C. to about 42° C. Thetemperature may be altered and higher temperatures used to replaceformamide and/or to give alternative stringency conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization, and at least about 0.5 M to atleast about 0.9 M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.01 M to at least about 0.15M salt for hybridization, and at least about 0.01 M to at least about0.15 M salt for washing conditions. In general, washing is carried outT_(m)=69.3+0.41 (G+C) % (Marmur and Doty, 1962). However, the T_(m) of aduplex DNA decreases by 1° C. with every increase of 1% in the number ofmismatch base pairs (Bonner and Laskey, 1974). Formamide is optional inthese hybridization conditions. Accordingly, particularly preferredlevels of stringency are defined as follows: low stringency is 6×SSCbuffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSCbuffer, 0.1% w/v SOS at a temperature in the range 20° C. to 65° C.;high stringency is 0.1×SSC buffer, 0.1% w/v SOS at a temperature of atleast 65° C.

The term “transformation” means alteration of the genotype of anorganism, for example, a eukaryotic cell, by the introduction of aforeign or endogenous nucleic acid.

By “vector” is meant a nucleic acid molecule, preferably a DNA moleculederived, for example, from a plasmid, bacteriophage, or plant virus,into which a nucleic acid sequence may be inserted or cloned. A vectorpreferably contains one or more unique restriction sites and may becapable of autonomous replication in a defined host cell including atarget cell or tissue or a progenitor cell or tissue thereof, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. Accordingly, the vector may be an autonomouslyreplicating vector, i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into a cell,is integrated into the genome of the recipient cell and replicatedtogether with the chromosome(s) into which it has been integrated. Avector system may comprise a single vector or plasmid., two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the cell into which the vector is to be introduced. The vector mayalso include a selection marker such as an antibiotic resistance genethat can be used for selection of suitable transformants. Examples ofsuch resistance genes are well known to those of skill in the art.

The term “gene” is used in its broadest sense and includes cDNAcorresponding to the exons of a gene. Accordingly, reference herein to a“gene” is to be taken to include:—

-   (i) a classical genomic gene consisting of transcriptional and/or    translational regulatory sequences and/or a coding region and/or    non-translated sequences (i.e. introns, 5′- and 3′-untranslated    sequences); or-   (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons)    and 5′- and 3′-untranslated sequences of the gene; and/or-   (iii) a structural region corresponding to the coding regions (i.e.    exons) optionally further comprising untranslated sequences and/or a    heterologous promoter sequence which consists of transcriptional    and/or translational regulatory regions capable of conferring    expression characteristics on said structural region.

The term “gene” is also used to describe synthetic or fusion moleculesencoding all or part of a functional product, in particular, a sense orantisense mRNA product or a peptide, oligopeptide or polypeptide or abiologically-active protein. Reference to a “gene” also includesreference to a “synthetic gene”.

The term “synthetic gene” refers to a non-naturally occurring gene ashereinbefore defined which preferably comprises at least one or moretranscriptional and/or translational regulatory sequences operablylinked to a structural gene sequence.

The term “structural gene” shall be taken to refer to a nucleotidesequence which is capable of being transmitted to produce mRNA andoptionally, encodes a peptide, oligopeptide, polypeptide or biologicallyactive protein molecule. Those skilled in the art will be aware that notall mRNA is capable of being translated into a peptide, oligopeptide,polypeptide or protein, for example, if the mRNA lacks a functionaltranslation start signal or alternatively, if the mRNA is antisensemRNA. The present invention clearly encompasses synthetic genescomprising nucleotide sequences which are not capable of encodingpeptides, oligopeptides, polypeptides or biologically-active proteins.In particular, the present inventors have found that such syntheticgenes may be advantageous in modifying target gene expression in cells,tissues or organs of a eukaryotic organism.

The term “structural gene region” refers to that part of a syntheticgene which is expressed in a cell, tissue or organ under the control ofa promoter sequence to which it is operably connected. A structural generegion may be operably under the control of a single promoter sequenceor multiple promoter sequences. Accordingly, the structural gene regionof a synthetic gene may comprise a nucleotide sequence which is capableof encoding an amino acid sequence or is complementary thereto. In thisregard, a structural gene region which is used in the performance of theinstant invention may also comprise a nucleotide sequence which encodesan amino acid sequence yet lacks a functional translation initiationcodon and/or a functional translation stop codon and, as a consequence,does not comprise a complete open reading frame. In the present context,the term “structural gene region” also extends to a non-codingnucleotide sequences, such as 5′-upstream or 3′-downstream sequences ofa gene which would not normally be translated in a eukaryotic cell whichexpresses said gene.

Accordingly, in the context of the present invention, a structural generegion may also comprise a fusion between two or more open readingframes of the same or different genes. In such embodiments, theinvention may be used to modulate the expression of one gene, bytargeting different non-contiguous regions thereof or alternatively, tosimultaneously modulate the expression of several different genes,including different genes of a multigene family. In the case of a fusionnucleic acid molecule which is non-endogenous to a eukaryotic cell andin particular comprises two or more nucleotide sequences derived from aviral pathogen, the fusion may provide the added advantage of conferringsimultaneous immunity or protection against several pathogens, bytargeting the expression of genes in said several pathogens.Alternatively or in addition, the fusion may provide more effectiveimmunity against any pathogen by targeting the expression of more thanone gene of that pathogen.

Particularly preferred structural gene regions according to this aspectof the invention are those which include at least one translatable openreading frame, more preferably further including a translational startcodon located at the 5′-end of said open reading frame, albeit notnecessarily at the 5′-terminus of said structural gene region. In thisregard, notwithstanding that the structural gene region may comprise atleast one translatable open reading frame and/or AUG or ATGtranslational start codon, the including of such sequences in no waysuggest that the present invention requires translation of theintroduced nucleic acid molecule to occur in order to modulate theexpression of the target gene. Whilst not being bound by any theory ormode of action, the inclusion of at least one translatable open readingframe and/or trauslational start codon in the subject nucleic acidmolecule may serve to increase stability of the mRNA transcriptionproduct thereof, thereby improving the efficiency of the invention.

The optimum number of structural gene sequences which may be involved inthe synthetic gene of the present invention will vary considerably,depending upon the length of each of said structural gene sequences,their orientation and degree of identity to each other. For example,those skilled in the art will be aware of the inherent instability ofpalindromic nucleotide sequences in vivo and the difficulties associatedwith constructing long synthetic genes comprising inverted repeatednucleotide sequences because of the tendency for such sequences torecombine in vivo. Notwithstanding such difficulties, the optimum numberof structural gene sequences to be included in the synthetic genes ofthe present invention may be determined empirically by those skilled inthe art, without any undue experimentation and by following standardprocedures such as the construction of the synthetic gene of theinvention using recombinase-deficient cell lines, reducing the number ofrepeated sequences to a level which eliminates or minimizesrecombination events and by keeping the total length of the multiplestructural gene sequence to an acceptable limit, preferably no more than5-10 kb, more preferably no more than 2-5 kb and even more preferably nomore than 0.5-2.0 kb in length.

For expression in eukaryotic cells, the construct generally comprises,in addition to the polynucleotide sequence, a promoter and optionallyother regulatory sequences designed to facilitate expression of thepolynucleotide sequence.

Reference herein to a “promoter” is to be taken in its broadest contextand includes the transcriptional regulatory sequences of a classicalgenomic gene, including the TATA box which is required for accuratetranscription initiation, with or without a CCAAT box sequence andadditional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or external stimuli, or in a tissue-specific manner. Apromoter is usually, but not necessarily, positioned upstream or 5′, ora structural gene region, the expression of which it regulates.Furthermore, the regulatory elements comprising a promoter are usuallypositioned within 2 kb of the start site of transcription of the gene.

In the present context, the term “promoter” is also used to describe asynthetic or fusion molecule, or derivative which confers, activates orenhances expression of a nucleic acid molecule in a cell.

Preferred promoters may contain additional copies of one or morespecific regulatory elements, to further enhance expression of the sensemolecule and/or to alter the spatial expression and/or temporalexpression of said sense molecule. For example, regulatory elementswhich confer copper inducibility may be placed adjacent to aheterologous promoter sequence driving expression of a sense molecule,thereby conferring copper inducibility on the expression of saidmolecules.

Placing a nucleic acid molecule under the regulatory control of apromoter sequence means positioning the said molecule such thatexpression is controlled by the promoter sequence. Promoters aregenerally positioned 5′ (upstream) to the genes that they control. Inthe construction of heterologous promoter/structural gene combinations,it is generally preferred to position the promoter at a distance fromthe gene transcription start site that is approximately the same as thedistance between that promoter and the gene it controls in its naturalsetting, i.e. the gene from which the promoter is derived. As is knownin the art, some variation in this distance can be accommodated withoutloss of promoter function. Similarly, the preferred positioning of aregulatory sequence element with respect to a heterologous gene to beplaced under its control is defined by the positioning of the element inits natural setting, i.e. the genes from which it is derived. Again, asis known in the art, some variation in this distance can also occur.

Examples of promoters suitable for use in the synthetic genes of thepresent invention include viral, fungal, bacterial, animal and plantderived promoters capable of functioning in plant, animal, insect,fungal, yeast or bacterial cells. The promoter may regulate theexpression of the structural gene component constitutively, ordifferentially with respect to cell, the tissue or organ in whichexpression occurs or, with respect to the developmental stage at whichexpression occurs, or in response to external stimuli such asphysiological stresses, or pathogens, or metal ions, amongst others.

Preferably, the promoter is capable of regulating expression of anucleic acid molecule in a eukaryotic cell, tissue or organ, at leastduring the period of time over which the target gene is expressedtherein and more preferably also immediately preceding the commencementof detectable expression of the target gene in said cell, tissue ororgan.

Accordingly, strong constitutive promoters are particularly useful forthe purposes of the present invention or promoters which may be inducedby virus infection or the commencement of target gene expression.

Plant-operable and animal-operable promoters are particularly preferredfor use in the construct of the present invention. Examples of preferredpromoters include the viral promoters such as bacteriophage T7 promoter,bacteriophage T3 promoter, SP6 promoter, bacterial promoters such as lacoperator-promoter, tac promoter, viral promotors such as SV40 latepromoter, SV40 early promoter, RSV-LTR promoter, CMV M promoter or plantviral promoters such as CaMV 35S promoter, SCSV promoter, SCBV promoterand the like.

In consideration of the preferred requirement for high-level expressionwhich coincides with expression of the target gene or precedesexpression of the target gene, it is highly desirable that the promotersequence is a constitutive strong promoter in the host of interest suchas the CMV-IE promoter or the SV40 early promoter sequence, the SV40late promoter sequence for mammalian cells and, the CaMV 35S promoter,or the SCBV promoter in certain plant cells, amongst others. Thosedrilled in the art will readily be aware of additional promotersequences other than those specifically described.

In the present context, the terms “in operable connection with” or“operably under the control” or similar shall be taken to indicate thatexpression of the structural gene region or multiple structural generegion is under the control of the promoter sequence with which it isspatially connected; in a cell, tissue, organ or whole organism.

The construct preferably contains additional regulatory elements forefficient transcription, for example, a transcription terminationsequence.

The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences generally containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active inplant cells are known and described in the literature. They may beisolated from bacteria, fungi, viruses, animals and/or plants orsynthesized de novo.

As with promoter sequences, the terminator may be any terminatorsequence which is operable in the cells, tissues or organs in which itis intended to be used.

Examples of terminators particularly suitable for use in the syntheticgenes of the present invention include the SV40 polyadenylation signal,the HSV TK polyadenylation the CYC1 terminator, ADH terminator, SPAterminator, nopaline synthase (NOS) gene terminator of Agrobacteriumtumefaciens, the terminator of the cauliflower mosaic virus (CaMV) 355gene, the zein gene terminator from Zea mays, the Rubisco small subunitgene (SSU) gene terminator sequences, subclover stunt virus (SCSV) genesequence terminators, any rho-independent E. coli terminator, or thelacZ alpha terminator, amongst others.

In a particularly preferred embodiment, the terminator is the SV40polyadenylation signal or the HSV TK polyadenylation signal which areoperable in animal cells, tissues and organs, octopine synthase (OCS) ornopaline synthase (NOS) terminator active in plant cells, tissue ororgans, or the lacZ alpha terminator which is active in prokaryoticcells.

Those skilled in the art will be aware of additional terminatorsequences which may be suitable for use in performing the invention.Such sequences may readily be used without any undue experimentation.

Means for introducing (i.e. transfecting or transforming) cells with theconstructs are well-known to those skilled in the art.

The constructs described supra are capable of being modified further,for example, by the inclusion of marker nucleotide sequences encoding adetectable marker enzyme or a functional analogue or derivative thereof,to facilitate detection of the synthetic gene in a cell, tissue or organin which it is expressed. According to this embodiment, the markernucleotide sequences will be present in a translatable format andexpressed, for example, as a fusion polypeptide with the translationproduct(s) of any one or more of the structural genes or alternativelyas a non-fusion polypeptide.

Those skilled in the art will be aware of how to produce the syntheticgenes described herein and of the requirements for obtaining theexpression thereof, when so desired, in a specific cell or cell-typeunder the conditions desired. In particular, it will be known to thoseskilled in the art that the genetic manipulations required to performthe present invention may require the propagation of a genetic constructdescribed herein or a derivative thereof in a prokaryotic cell such asan E. coli cell or a plant cell or an animal cell.

The constructs of the present invention may be introduced to a suitablecell, tissue or organ without modification as linear DNA, optionallycontained within a suitable carrier, such as a cell, virus particle orliposome, amongst others. To produce a genetic construct, the syntheticgene of the invention is inserted into a suitable vector or opisomemolecule, such as a bacteriophage vector, viral vector or a plasmid,cosmid or artificial chromosome vector which is capable of beingmaintained and/or replicated and/or expressed in the host cell, tissueor organ into which it is subsequently introduced.

Accordingly, a further aspect of the invention provides a geneticconstruct which at least comprises a genetic element as herein describedand one or more origins of replication and/or selectable marker genesequences.

Genetic constructs are particularly suitable for the transformation of aeukaryotic cell to introduce novel genetic traits thereto, in additionto the provision of resistance characteristics to viral pathogens. Suchadditional novel traits may be introduced in a separate geneticconstruct or, alternatively, on the same genetic construct whichcomprises the synthetic genes described herein. Those skilled in the artwill recognize the significant advantages, in particular in terms ofreduced genetic manipulations and tissue culture requirements andincreased cost-effectiveness, of including genetic sequences whichencode such additional traits and the synthetic genes described hereinin a single genetic construct.

Usually, an origin of replication or a selectable marker gene suitablefor use in bacteria is physically-separated from those genetic sequencescontained in the genetic construct which are intended to be expressed ortransferred to a eukaryotic cell, or integrated into the genome of aeukaryotic cell.

As used herein, the term “selectable marker gene” includes any genewhich confers a phenotype on a cell on which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with a genetic construct of the invention ora derivative thereof.

Suitable selectable marker genes contemplated herein include theampicillin-resistance gene (Amp^(r)), tetracycline-resistance gene(Tc^(r)), bacterial kanamycin-resistance gene (Kan^(r)), is the zeocinresistance gene (Zeocin is a drug of the bleomycin family which is trademark of InVitrogen Corporation), the AURI-C gene which confersresistance to the antibiotic aureobasidin A, phosphinothricin-resistancegene, neomycin phosphotransferase gen (nptII), hygromycin-resistancegene, β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase(CAT) gene, green fluorescent protein-encoding gene or the luciferasegene, amongst others.

Preferably, the selectable marker gene is the nptII gene or Kan^(r) geneor green fluorescent protein (GFP)-encoding gene.

Those skilled in the art will be aware of other selectable marker genesuseful in the performance of the present invention and the subjectinvention is not limited by the nature of the selectable marker gene.

The present invention extends to all genetic constructs essentially asdescribed herein, which include further genetic sequences intended forthe maintenance and/or replication of said genetic construct inprokaryotes or eukaryotes and/or the integration of said geneticconstruct or a part thereof into the genome of a eukaryotic cell ororganism.

Standard methods described supra may be used to introduce the constructsinto the cell, tissue or organ, for example, liposome-mediatedtransfection or transformation, transformation of cells with attenuatedvirus particles or bacterial cells, cell mating, transformation ortransfection procedures known to those skilled in the art.

Additional means for introducing recombinant DNA into plant tissue orcells include, but are not limited to, transformation using CaCl₂ andvariations thereof, direct DNA uptake into protoplasts, PEG-mediateduptake to protoplasts, microparticle bombardment, electroporation,microinjection of DNA, microparticle bombardment of tissue explant orcells, vacuum-infiltration of tissue with nucleic acid, or in the caseof plants, T-DNA-mediate transfer from Agrobacterium to the planttissue.

For microparticle bombardment of cells, a microparticle is propelledinto a cell to produce a transformed cell. Any suitable ballistic celltransformation methodology and apparatus can be used in performing thepresent invention. Exemplary apparatus and procedures are disclosed byStomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat.No. 4,945,050). When using ballistic transformation procedures, thegenetic construct may incorporate a plasmid capable of replicating inthe cell to be transformed.

Examples of microparticles suitable for use in such systems include 1 to5 μm gold spheres. The DNA construct may be deposited on themicroparticle by any suitable technique, such as by precipitation.

In a further embodiment of the present invention, the genetic constructsdescribed herein are adapted for integration into the genome of a cellin which it is expressed. Those skilled in the art will be aware that,in order to achieve integration of a genetic sequence or geneticconstruct into the genome of a host cell, certain additional geneticsequences may be required. In the case of plants, left and right bordersequences from the T-DNA of the Agrobacterium tumefaciens Ti plasmidwill generally be required.

The present invention further extends to an isolated cell, tissue ororgan comprising the constructs or parts thereof. The present inventionextends further to regenerated tissues, organs and whole organismsderived from said cells, tissues and organs and to propagules andprogeny thereof as well as seeds and other reproductive material.

For example, plants may be regenerated from transformed plant cells ortissues or organs on hormone-containing media and the regenerated plantsmay take a variety of forms, such as chimeras of transformed cells andnon-transformed cells; clonal transformants (e.g. all cells transformedto contain the expression cassette); grafts of transformed anduntransformed tissue (e.g. a transformed root stock grafted to anuntransformed scion in citrus species). Transformed plants may bepropagated by a variety of means, such as by clonal propagation orclassical breeding techniques. For example, a first generation (or T1)transformed plants may be selfed to give homozygous second generation(or T2) transformed plants, and the T2 plants further propagated throughclassical breeding techniques.

In another embodiment, the construct is used to induce modulation ofexpression of a target genetic sequence. For example, a construct, whenin linear form, comprises in the 5′ to 3′ direction an antisensesequence, a promoter and a sense sequence. These elements are thenflanked by viral-derived, replication-faciliating protein recognitionsequences (e.g. stem loop) or mammalian or microbial homologues. Aterminator sequence is located outside the recognition-sequence flankedregion. Upon replicative release, a polynucleotide sequence comprisingantisense and sense forms of a target genetic sequence is produced. Theresulting polynucleotide sequence may then form a hair-pin loop. Theconstruct may be varied to produce tandem or multiple repeats, invertsor combinations thereof. Such constructs are useful for gene silencingin plant and animal cells. The order of elements in the linear form isnot critical. For example, the location of the sense and antisensesequences may be exchanged. An example of one suitable construct isshown in FIG. 27.

The present invention is further described by the following non-limitingExamples.

Example 1 Expression Vectors Based on BBTV

A series of expression vectors were constructed which contain thecauliflower mosaic virus 35S promoter (355) driving expression of thegene encoding barnase, into which the intron from the potatolight-inducible tissue-specific ST-LS1 gene was introduced(NT-INTRON-CT). The terminator was derived from either the gene encodingnopaline synthase (nos) or the major open-reading frame (ORF) of BBTVDNA-6 (BT6).

The constructs were assembled using PCR with overlapping primers(Table 1) and cloned into pGEM-T vectors using standard techniques knownin the art. The expression vector pTBN was constructed and is shown inFIG. 1. pTBN (SEQ ID NO:66) represents the backbone upon which otherexpression vectors were constructed. Primer names and binding sites areindicated, as is the size of each sequence. pTBN (like the otherconstructs) was amplified in a step-wise manner. Initially, all threefragments were separately amplified (i.e. 35S, NT-INTRON-CT and nos).The entire sequence was then amplified by mixing each of the fragmentsin a PCR with primers +1 and −4.

pTBN6 (SEQ ID NO:67) shown in FIG. 2 contains a 624 by region containingthe CR-SL and CR-M of BBTV DNA-6 (61), inserted into the intron.

pTBN1 (SEQ ID NO:68) shown in FIG. 3 contains 163 by region containingthe CR-SL and CR-M of BBTV DNA-1 (1I), inserted into the intron,identically to pTBN6. The nos terminator was replaced by the 126 byterminator from BBTV DNA-6 large ORF.

Recircularization vectors are based upon pTBN6 and pTBN1. They areflanked by Rep recognition sequences and designed to recircularize andsubsequently be transcriptionally active, only in the presence of theBBTV Rep. Three different vectors were made: pRTBN6, pRTBN1 andpRTBN1/6.

pRTBN6 shown in FIG. 4 was constructed from pTBN6. Two fragments wereamplified using +5 and −4 and +1 and −6 respectively. These were clonedinto pGEM-T and sub-cloned to create pRTBN6.

pRTBN1 shown in FIG. 5 was constructed from pTBN1 in a similar fashionto pRTBN6.

pRTBN1/6 shown in FIG. 6 was a hybrid of pRTBN1 and pRTBN6.

Untranslatable vectors were also constructed for each of the constructsmentioned above (except pTBN). In these vectors, the start codon of thebarnase gene was deleted using the +11 primer (refer to Table 1). Theconstructs were named pUBN6, pUBN1, pRUBN6 and pRUBN1.

TABLE 1 Oligonucleotide primer sequences Primer Name  Sequence (5′-3′)+1 3TS1-H AAGCTTCATGGAGTCAAAGA  (SEQ ID NO: 1) +2 35S1-BTCATTTGGAGAGGATCCATGGCACAGGTT  (SEQ ID NO: 2) −2 B2-35SAACCTGTGCCATGGATCCTCTCCAAATGA  (SEQ ID NO: 3) +3 B1-NAAAATCAGATAAGAGCTCGATCGTTCAAA  (SEQ ID NO: 4) −3 N2-BTTTGAACGATCGAGCTCTTATCTGATTTT  (SEQ ID NO: 5) −4 NOS4-HAAGCTTTTCGCCATTCAGGCTGC  (SEQ ID NO: 6) +5 B3-6TATCATTAATTAGTAAGTTGTGCTGTAA  (SEQ ID NO: 7) −5 6-B4TTACAGCACAACTTACTAATTAATGATA  (SEQ ID NO: 8) +6 6-B3GGAAGGCAGAAGCGAGTAATATAATATT  (SEQ ID NO: 9) −6 B4-6AATATTATATTACTCGCTTCTGCCTTCC  (SEQ ID NO: 10) +7 B5-IATCATTAATTAGTCACACTATGACAAAAG  (SEQ ID NO: 11) −7 I-B6TTGTCATAGTGTGACTAATTAATGATAAT  (SEQ ID NO: 12) +8 I-B5GACATTTGCATCAGTAATATAATATTTCA  (SEQ ID NO: 13) −8 B6-IAATATTATATTACTGATGCAAATGTCCCG  (SEQ ID NO: 14) +9 B7-6TTCAGATAAGAGCTCAGTAACAGCAACAAC  (SEQ ID NO: 15) −9 6T2-BGCTGTTACTGAGCTCTTATCTGATCTTTG  (SEQ ID NO: 16) −10 6T4-HAAGCTTATTTCCCAAATATACGT  (SEQ ID NO: 17) +11 UNTBarnGGATCCGCACAGGTTATCAAC  (SEQ ID NO: 18)

Example 2 Expression Vectors Based on TYDV (a) Construction of anIntron-Containing Gus Reporter Gene Expression Cassette

The vector pCAMBIA 2301 was obtained from CAMBIA (Can berra, Australia).This vector contains a 189 by catalase intron within the 5′ portion ofthe uidA coding region. The CaMV 35S promoter region (800 bp), uidAcoding region, and nos terminator were removed from pCAMBIA 2301 as aHindIII/SphI fragment and inserted into similarly digested pGEM-T(Promega) vector. The subsequent construct was designated pGEM-2301. The800 by CaMV 35S promoter was replaced with the stronger 530 by CaMV 35Spromoter by NotI/BglII digestion and ligation. The subsequent vector wasdesignated p35S-2301 (FIG. 7) and served as the template for allsubsequent cloning steps.

(b) Isolation of the Tobacco Yellow Dwarf Mastrevirus (TYDV) 9 LargeIntergenic Region and Insertion into the Catalase Intron of p35S-2301

A 272 by fragment incorporating the large intergenic region (LIR) (nt +1to nt +272) of TYDV (Genbank Ace M81103) was amplified fromTYDV-infected tobacco leaf tissue by PCR using primers LIR-F and LIR-R(see FIG. 1). This fragment was designated LIR.

Primers:

LIR-F [SEQ ID NO: 19] 5′-GCTCTTCCTGCAGGCGGCCGCATTAAGGCTCAAGTACCGTA 3′LIR-R [SEQ ID NO: 20] 5′-GCTCTTCGTCGACGAATTCATTTTCAACTTTGGGATGTCAC-3′

The segment comprising the CaMV 35S promoter (530 bp), uidA 1^(st) exon(19 bp), and 5′ half of the catalase intron (83 bp) was amplified fromp35S-2301 plasmid DNA by PCR, using primers 35S-IE and CAT-A. Thisfragment was designated CAT-A.

Primers:

[SEQ ID NO: 21] 35S-IE 5′-GAATTCCATGGAGTCAAAGATTCA-3′ [SEQ ID NO: 22]CAT-A 5′-GCCCGCTGCAGAGTTTAAAGAAAGATCAAAGC-3′

The segment comprising the 3′ half of the catalase intron (106 bp) anduidA 2^(nd) exon (1809 bp) was amplified from p35S-2301 plasmid DNA byPCR, using primers CAT-B and GUS-BstEII. This fragment was designatedCAT-B.

Primers:

CAT-B [SEQ ID NO: 23] 5′-GCCCCGGTCGACGATCTATTTTTTAATTGATTGG-3′GUS-BstEII [SEQ ID NO: 24] 5′-TTCGAGCTGGTCACCTGTAATTCACACGTGGTG-3′

The resulting PCR products were cloned into pGEM-T and the nucleotidesequence verified. Ultimately, each fragment was excised (CAT-A usingEcoRI/PstI, LIR using PstI/SalI, and CAT-B using SalI/BstEII) frompGEM-T, and ligated together into EcoRI/BstEII digested p35S-2301 tocreate the plasmid pTEST1 (FIG. 8).

(c) To Determine Whether GUS Expression is Affected by Insertion of theTYDV LIR into the Catalase Intron

In order to determine whether GUS expression, and therefore intronsplicing, was affected by insertion of the TYDV LIR into the catalaseintron of p355-2301, constructs were bombarded into embryogenic bananacells and GUS activity transiently assayed. Test plasmid pTEST1 andpositive control plasmid p35S-2301 were coated onto 1 μm gold particlesand biolistically introduced into 5 day old banana (Musa spp. cv.“Ladyfinger” AAA) embryogenic cells according to Becker et al. (2000).Two days post-bombardment, cells were harvested and GUS activity assayedhistochemically (Jefferson et al., 1987).

No endogenous GUS activity was observed in non-bombarded cells. StrongGUS activity, evident as bright blue staining cell foci, was observedfrom cells bombarded with the positive control plasmid p355-2301. Incontrast, GUS expression from cells bombarded with pTEST1, was lower(about 5-fold) as determined by number and intensity of blue stainingcell foci. This result suggested that insertion of the TYDV LIR into thecatalase intron of p358-2301 does not abolish GUS expression, but mayaffect intron processing to some degree.

(d) Identification of Cryptic Intron Splice Sites within the TYDV LIR

In order to determine whether the TYDV LIR contained potential crypticintron splice sites, which may affect pre-mRNA processing, cDNA wassynthesized from RNA extracts derived from cells bombarded withp355-2301 and pTEST1. Total RNA was isolated from banana cells two dayspost bombardment with p355-2301 and pTEST1 using the method of Chang etal. (1993) Complementary DNA was synthesized from total RNA using theprimer uidA2. This cDNA served as a template for a nested PCR usingprimers uidA1 and uidA3. The resulting PCR products were cloned intopGEM-T and sequenced. Sequencing identified two potential sites withinthe TYDV LIR, which may contribute to aberrant splicing of the catalasebaton from the uidA coding region pre-mRNA. The first sequence,CTGCAGVGC, located within the primer LIR-F used to isolated the TYDVLIR, bears strong similarity to the consensus 3′ splice site(T(10X)GCAG∇GT). The second sequence, TA∇GTGAGT (nt +43 to nt +50),shares some similarity to the consensus 5′ splice site (AG∇GTAAGT).

Primers:

uidA1 5′-CCATGGTAGATCTGAGGG-3′ [SEQ ID NO: 25] uidA25′-TACGTACACTTTTCCCGGCAATAAC-3′ [SEQ ID NO: 26] uidA35′-GTAACGCGCTTTCCCACCAACGC-3′ [SEQ ID NO: 27](e) Removal of the 3′ Cryptic Intron Splice Site from the TYDV LIR

Of the two cryptic intron splice sites identified, the first (CTGCAGGC)was considered the most significant due to its location in relation tothe 5′ catalase intron splice site. In order to remove this sequencefrom the TYDV LIR, a new primer, LIR-Xho, was designed incorporating aXhoI site in place of the original PstI and NotI restriction sites.

The TYDV LIR was re-amplified from pTEST1 plasmid DNA by PCR usingprimers LIR-Xho and LIR-R. This fragment was designated LIR-X.Similarly, the fragment comprising the CaMV 35S promoter (530 bp), uidA1^(st) exon (19 bp) and 5′ half of the catalase intron (83 bp) wasre-amplified from pTEST1 plasmid DNA by PCR using primers FUP andCAT-Xho. This fragment was designated CAT-X. Both PCR products werecloned into pGEM-T and their sequences verified. Ultimately, PCRfragments were excised from pGEM-T (LIR-X using XhoI/SalI and CAT-Xusing PstI/XhoI) and ligated into PstI/SalI digested pTEST1, to replacethe original inserts. This construct was designated pTEST2 (FIG. 9).Removal of the cryptic intron splice site in pTEST2 generated higherlevels of GUS expression than pTEST1 due to a reduction in aberrantsplicing and improved mRNA processing.

Primers:

LIR-Xho 5′-CTCGAGATTAAGGCTCAAGTACCGTA-3′ [SEQ ID NO: 28] CAT-Xho5′-AGTTTAAAGAAAGATCAAAGC-3′ [SEQ ID NO: 29] FUP5′-AATTAACCCTCACTAAAGGG-3′ [SEQ ID NO: 30]

(f) Construction of the Rep-Activatable GUS Expression Vector

A 229 by fragment incorporating the TYDV small intergenic region (SIR)(nt +1275 to nt +1504) was amplified from TYDV-infected tobacco leaftissue by PCR using primers SIR-F and SIR-R (see FIG. 2). The resultingPCR product was cloned into pGEM-T and the nucleotide sequence verified.This plasmid was designated pGEM-SIR. The TYDV SIR was excised frompGEM-SIR as a SphI fragment and inserted into the unique SphI site,downstream of the nos terminator, in pTEST1. This construct wasdesignated pTEST1-SIR.

Primers:

SIR-F [SEQ ID NO: 31] 5′-GCATGCAAGAGTTGGCGGTAGATTCCGCATGT-3′ SIR-R[SEQ ID NO: 32] 5′-GCTCTTCGCGGCCGCGCTCCTGAATCGTCGAGTCA-3′

The CaMV 35S promoter, uidA exon, catalase intron 5′ half, and TYDV LIRwere excised from pTEST2 as a NotI/SalI fragment and inserted into asimilarly-digested pGEM-T vector. The subsequent clone was designatedpGEM-CATX. The TYDV catalase intron 3′ half, uidA 2^(nd) exon, nosterminator, and TYDV SIR were excised from pTEST1-SIR as a NotI fragmentand inserted into the unique NotI site in pGEM-CATX. This construct wasdesignated pTEST3 (FIG. 10). The activatable GUS expression cassette wassubsequently excised from pTEST3 by SacI/ApaI digestion, and insertedinto the SacI/ApaI restriction sites located upstream of the nospro-NPTII-nos ter cassette in the binary plasmid pART27 (cleave 1992).This construct was designated pTEST4 (FIG. 11). The vector pTEST4 wasintroduced into Agrobacterium tumefaciens (LBA4404) by electroporationusing the method of Singh et al. (1993).

(g) Construction of the Infectious TYDV 1.1mer

Two overlapping fragments of the TYDV genome were amplified fromTYDV-infected tobacco leaf tissue by PCR using primer pairs LIR-F/SIR-R(SEQ ID NO:19/SEQ ID NO:32) and LIR-R/TYD-3F (SEQ ID NO:20/SEQ IDNO:33). The resulting PCR products (TYD-R and TYD-L, respectively) werecloned into pGEM-T and their sequences verified. These plasmids weredesignated pGEM-TYD-R and pGEM-TYD-L, respectively. A 1659 by fragmentof the TYDV genome was excised from pGEM-TYD-L by digestion with EcoRI,and inserted into the unique EcoRI site in pGEM-TYD-R. This constructwas designated pGEM-TYDV1.1mer. The TYDV 1.1mer was excised frompGEM-TYDV1.1mer by SalI/EcoRI partial digestion and inserted intosimilarly digested pBI101.3 vector (Clontech), to replace the uidA geneand nos terminator. This construct was designated pBI-TYDV1.1mer (FIG.12). The vector pEI-TYDV1.1mer was introduced into Agrobacteriumtumefaciens (LBA4404) by electroporation using the method of Singh etal. (1993).

Primers:

TYD-3F 5′-TTTAAACGTTTAGGGGTTAGCA-3′ [SEQ ID NO: 33]

(h) Construction of the CaMV 35S-TYDV Rep Fusion

The complete Rep (including RepA) gene of TYDV (nt +2580 to nt +1481)was amplified from TYDV-infected tobacco leaf tissue by PCR usingprimers TYDVRepF and TYDVRepR. The resulting PCR product was directlycloned into the SmaI site located between the CaMV 35S promoter (530 bp)and CaMV 35S terminator (200 bp) in pDH51 (Pietrzak et al., 1986). Thisconstruct was designated p35S-Rep (FIG. 13).

Primers:

TYDVRepF 5′-TCAGTGACTCGACGATTC-3′ [SEQ ID NO: 34] TYDVRepR5′-TTAATATGCCTTCAGCCC-3′ [SEQ ID NO: 35]

Example 3 GUS Expression Assays Using TYDV Vectors (a) TransientRep-Activated Expression of GUS in Dicot and Monocot Cells

Tobacco (NT-1) cells are maintained essentially as described by An(1985), and prepared for microparticle bombardment as detailed byDugdale et al. (1998). Banana (Musa spp. Cv. “Ladyfinger” AAA)embryogenic cell suspensions were prepared as previously described.Coating of gold particles and biolistic parameters were essentially asdescribed by Dugdale et al. (1998) or Becker et al. (2000).

Plasmids used for this study included:—

(i) p355-2301 as positive control (FIG. 7),(ii) pTEST3 (FIG. 10),(iii) p355-Rep (FIG. 13), and(iv) pTEST3 and p35S-Rep.

Five plates of both cell lines are bombarded for each of the fourplasmid combinations. Cells are harvested three days post-bombardmentand GUS activity assayed histochemically and/or fluorometrically(Jefferson et al., 1987).

No endogenous GUS activity is observed in non-bombarded cells. StrongGUS activity, evident as bright blue staining cell foci, is observedfrom cells bombarded with the positive control plasmid p35S-2301. No GUSexpression is observed from cells bombarded with either p35S-Rep orpTEST3. In contrast, cells bombarded with both p355-Rep and pTEST3 stainintensely blue, greater than that obtained with the positive controlplasmid p358-2301. This result suggests that only upon addition of theTYDV Rep in trans does the GUS expression cassette in pTEST3 becomeactivated.

(b) Detection of Rep-Assisted Nicking, Joining and Replication of theGUS Multicopy Plant Episome (MPE)

Detection of the TYDV-based GUS MPEs is achieved using a PCR approach.Primers uidA1 (SEQ ID NO:25) and uidA3 (SEQ ID NO:27) amplify a fragmentof the uidA gene spanning the catalase intron. Only upon Rep-assistedrelease of the TYDV-based MPE from the plasmid pTEST3 does this primercombination generate a 600 by product (including 140 by of the uidAgene, 190 by of the catalase intron and 270 by of the TYDV LIR) in aPCR.

Tobacco NT-1 and banana cells are bombarded with each of the fourplasmid combinations listed above. Three days post-bombardment, cellsare harvested and total gDNA extracted using the method of Stewart andVia (1993). Total gDNA (1 μg) is used as a template for a PCR withprimers uidA1 (SEQ ID NO:25) and uidA3 (SEQ ID NO:27). PCR products areelectrophoresed through a 1.5% w/v agarose gel. A 330 by product isobtained from gDNA of cells bombarded with the control plasmid p358-2301(FIG. 7). This product corresponded to the uidA and catalase intronsequence from the input plasmid DNA. No PCR product is obtained fromgDNA of non-bombarded cells, or cells bombarded with pTEST3 or p35S-Repalone. A 600 by product is obtained from gDNA of cells bombarded withboth pTEST3 and p35S-Rep. This result supports previous GUShistochemical assays and suggests the TYDV-based MPEs are only generatedin cells bombarded with both pTEST3 and p358-Rep.

Replication of the TYDV-based MPEs is assessed by Southernhybridization. Using this approach, multimeric forms of the MPE,indicative of rolling circle replication, are detected by hybridisation.Total gDNA (20 ug) from cells bombarded with each of the four plasmidcombinations, are electrophoresed through a 1.5% w/v agarose gel. DNA istransferred to a nylon membrane (Roche) by the method of Southern(1975). A 600 by DIG-labelled probe, specific for the uidA and catalaseintron, is amplified by PCR using primers uidA1 (SEQ ID NO:25) and uidA3(SEQ ID NO:27). The uidA-specific probe is hybridised with the nylonmembrane at 42° C. in DIG Easy-Hyb solution (Roche) and signal detectedusing CDP-Star substrate (Roche) according to manufacturer'sinstructions. Characteristic supercoiled, linear, and open circularforms and higher molecular weight multimeric forms of the TYVD-basedMPEs are only detected in gDNA from plant cells bombarded with bothpTEST3 and p35S-2301. Together these results confirm that, when providedin trans, the TYDV Rep is capable of nicking, joining, and replicatingthe TYDV-based AVE in both monocotyledonous and dicotyledonous celltypes. Further, these results suggest that uidA expression from theplasmid pTEST3 is only activated upon addition of the TYDV Rep, and theaddition results in significantly higher expression than anon-replicating GUS expression cassette (p35S-2301).

Example 4 Stable Transformation of a Monocotyledonous and aDicotyledonous Plant with the Rep-Activatable Cassette

Banana (Musa spp. Cv. “Ladyfinger” AAA) embryogenic cell suspensions aretargeted for microprojectile-based stable transformation. Cells arebombarded with pTEST3, as previously described, except the plasmid isco-transformed with 1 ug of pDHKAN (Pietrzak et al., 1986). This plasmidcontains a CaMV 35S pro-NPTII-CaMV 355 ter cassette, from whichexpression of the NPTII gene confers resistance to the antibioticskanamycin or geneticin. Selection, culturing and regeneration oftransgenic banana plants are done essentially as described by Becker etal. (2000). Independent transgenic plants are confirmed to contain boththe NPTII and uidA genes by PCR, using primer pairs NPT-F/NPT-R anduidA4/uidA5, respectively. Ten independent transformants are selectedfor further studies.

Primers:

NPT-F 5′-ATGATTGAACAAGATGGATT-3′ [SEQ ID NO: 36] NPT-R5′-TGAGAAGAACTCGTCAAGA-3′ [SEQ ID NO: 37] uidA45′-GTTATTGCCGGGAAAAGTGTACGTA-3′ [SEQ ID NO: 38] uidA55′-CTAGCTTGTTTGCCTCCCTGCTGCG-3′ [SEQ ID NO: 39]

Tobacco (Nicatiana tabacum cv. “Samsun”) is transformed byAgrobacterium-mediated infection of leaf discs according to the methodof Horsch et al. (1988). Ten independent transgenic plants aretransformed with T-DNA from the plasmid pTEST4. Each line is shown tocontain the NPTII and uidA coding regions by PCR, as described above.

Leaf pieces from each of the ten transgenic banana and tobacco linestransformed with the Rep-activatable GUS expression cassette (i.e.pTEST3 [FIG. 10] and pTEST4 [FIG. 11], respectively) are bombarded withthe plasmid p355-Rep. Three days post-bombardment, leaf pieces aresubjected to GUS histochemical assays. No GUS expression is evident inunshot leaf pieces from each of the ten banana and tobacco lines. Leafpieces, bombarded with the plasmid p35S-Rep, display multiple blueGUS-staining foci. Rep-directed nicking, joining, and replication of theTYDV based MPEs is confirmed in these leaf pieces, as describedpreviously. These results indicate the TYDV Rep is capable of activatingGUS expression from a stably integrated copy of either plasmid, and ableto nick, join and replicate the TYDV-based MPE in vivo.

Example 5 TYDV-Infection Activated Expression in Transgenic Tobacco

Each of the 10 transgenic tobacco lines is infiltrated withAgrobacterium cultures transformed with pBI-TYDV1.1mer (refer to Example2(f), FIG. 12) using the method of Boulton (1995). Over a two monthperiod, samples are taken from the point of infection and throughout theplant, and GUS expression assessed using histochemical assays. GUSactivity (i.e. blue staining tissue) is only noted in TYDV-infectedplants, compared to mock-inoculated controls. Over time, GUS expressionspreads, via the vasculature, from the initial point of infection tovarious plant parts. Rep-directed nicking, joining and replication ofthe GUS expression cassette is established as previously described. Thisresult suggests that TYDV infection is sufficient for replicativerelease of the GUS expression cassette from an integrated chromosomalcopy.

Example 6 Transient Cell-Death Assays Using Expression Vectors Based onBBTV

To demonstrate that barnase was capable of causing cell death, assayswere carried out with the expression vectors (pTBN, pTBN6, pTBN1, FIGS.1, 2 and 3). Negative controls were pUBN6 and pUBN1 (refer to Example 1,above). Each of these constructs was co-bombarded with a GUS vector intobanana (Musa spp cv. Bluggoe) embryogenic cell suspensions essentiallyas described by Dugdale et al, 1998. The GUS vector contained a strongpromoter (maize Ubi1, CaMV 35S or banana Act1 driving the expression ofthe reporter gene β-glucuronidase (Jefferson, 1987). Subsequent MUGassays for GUS activity showed that cells which were transformed witheither pTBN, pTBN6 or pTBN1 had lower GUS activity than did the negativecontrol (pUBN1 or pUBN6) (FIG. 14). This suggests that intron splicingis still occurring, and at least in the case of pTBN1, does not differsignificantly from the original pTBN vector. Thus, the inclusion of theBBTV replication elements into the intron did not significantly decreasethe splicing efficiency or subsequent activity of barnase, relative topTBN.

Experiments were conducted that showed that recircularization andreplication of BBTV based “1.1 mers” occurs in the presence of the Rep(gene product from BBTV DNA-1). It was also found that replication wasenhanced by inclusion of the gene product of BBTV DNA-5 (a putativeretinoblastoma binding-like protein). Consequently, each of therecircularization vectors (pRTBN6 [FIG. 4], pRTBN1 [FIG. 5], pRUBN6,pRUBN1) was bombarded with BBTV DNA-1 and 5 “1.1 mers” and a GUSexpression vector.

TABLE 2 Plasmid combinations used for microprojectile bombardment ofbanana cells x-axis label (FIG. 15) RTBN6 RUBN6 RTBN1 RUBN1 BBTV1 ✓ ✓ ✓✓ BBTV5 ✓ ✓ ✓ ✓ GUS ✓ ✓ ✓ ✓ RTBN6 ✓ — — — RUBN6 — ✓ — — RTBN1 — — ✓ —RUBN1 — — — ✓

The results shown in FIG. 15 supported the previous expression vectorcell death assays (FIG. 14). Again, the constructs containinguntranslatable barnase (pRUBN6 and pRUBN1) had higher GUS activity thanthe translatable constructs.

Experiments were conducted to demonstrate that barnase activity wasinduced only in the presence of the Rep protein. Consequently, assayswere carried out+/−the Rep (BBTV DNA-1 “1.1 mer”) to demonstrate thatexpression would only occur when it was present (Table 3). “Stuffer” DNAwas used to keep a constant DNA concentration

TABLE 3 Plasmid combinations for microprojectile bombardment of bananacells X-axis label (FIG. 16) RTBN6+ RTBN6− RUBN6+ RTBN1+ RTBN1− RUBN1+BBTV1 ✓ — ✓ ✓ — ✓ BBTV5 ✓ ✓ ✓ ✓ ✓ ✓ GUS ✓ ✓ ✓ ✓ ✓ ✓ RTBN6 ✓ ✓ — — — —RUBN6 — — ✓ — — — RTBN1 — — — ✓ ✓ — RUBN1 — — — — — ✓ Stuffer — ✓ — — ✓—

To observe if the recircularization constructs were able to replicate inthe presence of BBTV DNA-1 and 5 “1.1 mers”, untranslatable constructswere included in transient banana cell replication assays. Cells wereharvested at 0, 4 and 8 days after bombardment, total cellular DNAextracted and analyzed using Southern hybridization.

Initially, the membranes were probed with a DIG-labelled CaMV 358 probe.No replicative forms were evident in cells at day 4 or 8. However, atday 0, potential replicative forms were present in very lowconcentrations in both pRUBN6 and pURBN1. The 35S probe was stripped andthe membranes reprobed with a DIG-labelled BBTV DNA-1 probe. High levelsof replication were observed in cells harvested on both day 4 and clay8, and almost none in cells harvested on day 0.

Example 7 Cell-Death Assays Using Expression Vectors Based on TYDV (a) ARep-Activatable Suicide Gene Vector to Confer Resistance to TYDV

The plasmid pRTBN (DNA Plant Technologies, Oakland, Calif.). containsthe barnase coding region (339 bp) within which has been incorporatedthe potato ST LS1 intron (188 bp). The entire barnase gene and intronwas amplified from pRTBN by PCR using primers BARN.EXP1 and BARN.EXP2.An untranslatable gene control was similarly amplified using primersBARN.UTR and BARN.EXP2.

Primers:

BARN.EXP1 [SEQ ID NO: 40] 5′-GGATCCATGGCACAGGTTATCAACACGTTTGACG-3′BARN.EXP2 [SEQ ID NO: 41] 5′-CTAGAGTTATCTGATTTTTGTAAAGGTC-3′ BARN.UTR[SEQ ID NO: 42] 5′-GGATCCGCACAGGTTATCAACACGTTTGACG-3′

PCR products were cloned into pGEM-T vector. These clones weredesignated pGEM-BTR and pGEM-BUTR, respectively. The TYDV LIR (LIR-X)was excised as an EcoRI fragment from pGEM-T and inserted into the MfeIsite located within the potato LTS intron of pGEM-BTR and pGEM-BUTR.These plasmids were designated pBTR-LIR and pBUTR-LIR, respectively. TheLIR-containing barnase genes in pBTR-LIR and pBUTR-LIR were excised asBamHI/PstI fragments and inserted into similarly-digested pGUS2 vectorto replace the original uidA coding region. Plasmid pGUS2 contains aCaMV35S pro (530 bp)-uidA gene-CaMV 35S ter (200 bp). These constructswere designated p35S-BTR-LTR and p35S-BUTR-LIR, respectively (FIG. 17).Two control plasmids were constructed by excision of the bamase genesfrom pGEM-BTR and pGEM-BUTR with BamHI/PstI, and insertion intosimilarly digested pGUS2. These control plasmids were designatedp35S-BTR and p35S-BUTR, respectively (FIG. 18).

(b) Transient Assessment of TYDV Rep-Activated Barnase Activity inMonocotyledonous and Dicotyledonous Cells

In order to determine barnase activity in vivo, suicide constructs areco-bombarded with a green fluorescent protein (gfp) expression cassette.Barnase expression and action (i.e. cell death) is considered to occurwhen a significant reduction in green fluorescent foci is observed incomparison to the untranslatable barnase controls.

Banana (Musa spp. Cv. “Ladyfinger”) and tobacco (Nicotiana tabacum NT-1)cells are bombarded with plasmids (i) p35S-BTR, (ii) p35S-BUTR, (iii)p35S-BTR-LIR, and (iv) p35S-BUTR-LIR (FIGS. 17 and 18) as described inExample 3, above. Each plasmid is co-bombarded with 1 ug of pWORM. Theconstruct pWORM contains a CaMV 355 pro (530 bp)-gfp (750 bp)-CaMV 35Ster (200 bp) cassette and has previously been shown to provide stronggreen fluorescence in transient assays with both cell types (Dugdale etal., 1998).

Three days post-bombardment, green fluorescence is visualised using aLeica MZ12 stereo microscope with GFP-Plus fluorescence module(excitation=490, emission=510). Both p35S-BTR and p35S-BTR-LIRsignificantly reduce gfp expression from pWORM (as determined by thenumber and intensity of green fluorescent foci) in comparison top355-BUTR and p35S-BUTR-LIR. This result suggests that insertion of theTYDV LIR into the ST LS1 intron within p355-BTR does not interfere withintron processing nor inhibit barnase expression.

(c) Construction of the TYDV Rep-Activatable Barnase Vector

The CaMV 35S promoter, barnase 5′ gene half, ST LS1 5′ intron half andTYDV LIR are re-amplified from p35S-BTR-LIR (FIG. 17A) by PCR usingprimers 35S-IE (SEQ ID NO:21) and LIR-R (SEQ ID NO:20). The PCR productis cloned into pGEM-T vector and sequence-verified. This plasmid isdesignated pGEMB5′. The TYDV LIR, ST LS1 3′ intron half, barnase 3′ genehalf and nos terminator is excised from p35S-BTR-LIR as a XhoI/SacIfragment, the TYDV SIR is excised from pGEM-SIR as a SacI/NcoI fragment,and the CaMV 35S promoter, barnase 5′ gene half, ST LS1 5′ intron halfand TYDV LIR are excised from pGEMB5′ as a NcoI/SacII partial fragment.Inserts are ligated together with XhoI/SacII digested pBluescript II(Stratagene). The resulting construct is designated pBTR.test1 (FIG.19A). The untranslatable control vector is similarly prepared and theresulting construct designated pBUTR.test1 (FIG. 19B).

(d) Transient TYDV Rep-Activated Barnase Expression in Monocot and DicotCells.

Banana (Musa spp. Cv. “Ladyfinger”) and tobacco (Nicotiana tabacum NT-1)cells are bombarded, as described above, with the plasmid combinationslisted in Table 4, below:—

TABLE 4 Plasmid combinations for transient transformation assays inbanana and tobacco cells, and there resulting gfp expression (asssessedas + or −) Plasmid combination pBTR-test1 pBUTR-test1 p35S-BTR p35S-BUTRpBTR-test1 pBUTR-test1 pWORM pWORM pWORM pWORM pWORM pWORM p35S-Repp35S-Rep Green fluorescent no yes yes yes no yes foci 3 days post-bombardment

Results in Table 4 suggest that barnase expression (and therefore celldeath) is only activated from pBTR-test1 when the TYDV Rep is suppliedin trans. Rep-activated expression of the untranslatable barnase genecassette (pBUTR-test1) results in no significant reduction in gfpexpression from pWORM. Rep-assisted nicking, joining, and replication ofthe MPEs from cells bombarded with pBUTR-test1, pWORM, and p35S-Rep isconfirmed as previously described, except primers BARN.UTR (SEQ IDNO:40) and BARN.EXP2 (SEQ ID NO:41) are used for PCR and a DIG-labelledbarnase-specific probe is synthesised using the before-mentionedprimers.

(e) Construction of Binary Plasmids Containing the Rep-ActivatableBarnase Gene Cassettes

Rep-activatable barnase cassettes are excised from pBTR-test1 andpBUTR-test1 as PvuII fragments and inserted into the unique EcoRI site(blunt ended using DNA polymerase I large Klenow fragment) locateddownstream of the CaMV 35S pro-NPTII-CaMV 35S ter cassette in the binaryplasmid pTAB5 (CSIRO, Canberra, Australia). The resulting constructs aredesignated pTAB-BTR1 and pTAB-BUTR1, respectively. Both vectors areintroduced into Agrobacterium tumefaciens (LBA4404) by electroporationusing the method of Singh et al. (1993).

(f) Stable Transformation of a Monocotyledonous and a DicotyledonousPlant with the Rep-Activatable Barnase Cassettes.

Stable transformation of banana (Musa spp. Cv. “Ladyfinger”) and tobacco(Nicotiana tabacum cv. “Samsun”) is done as described in Example 4,above, except plasmids pBTR-test1 and pBUTR-test1 are independentlyco-transformed with pDHKAN for stable banana transformation andAgrobacterium cultures harbouring the plasmids pTAB-BTR1 and pTAB-BUTR1are used for Agrobacterium-mediated transformation of tobacco leafdisks.

Transformed plants are confirmed to contain the barnase gene cassettesand the NPTII gene by PCR using primer pairs LIR-F/LIR-R (SEQ IDNO:19/SEQ ID NO:20) and NPT-F/NPT-R (SEQ ID NO:36/SEQ ID NO:37),respectively. Ten independent transgenic lines of both monocotyl-edonousand dicotyledonous species are selected for further studies.

(g) Rep-Activated Hypersensitive Resistance to TYDV in TransgenicTobacco

Rep-activation of barnase expression in the ten independent tobaccoplants transformed with pTAB-BTR1 and pTAB-BUTR1 is initially tested byparticle bombardment of the p35S-Rep construct into leaf pieces, asdescribed. Two days post-bombardment, necrosis of bombarded areas isonly evident on leaves of tobacco plants transformed with theRep-activatable translatable barnase gene cassette (pTAB-BTR1) incomparison to the untranslatable control (pTAB-BUTR-1). This resultsuggests introduction of the TYDV Rep in trans is sufficient forreplicative release of the barnase expression cassette from anintegrated chromosomal copy. Rep-assisted nicking, joining, andreplication is confirmed in leaf pieces of tobacco plants transformedwith pTAB-BUTR1, as was described for the transient assays in NT-1cells.

To demonstrate hypersensitive resistance to TYDV in transgenic tobacco,viruliferous leafhoppers (Orosius argentatus) are allowed to feed onplants for up to 2 days. Over the following week, plants are inspectedfor characteristic TYDV symptoms as described originally by Hill (1937).Plants transformed with the Rep-activatable, untranslatable barnaseexpression cassette (pTAB-BUTR1) produce typical TYDV symptoms,including dwarfing, yellowing, bending down of margins and tips of youngleaves, and shortening of the internodes. In contrast, tobacco plantstransformed with the Rep-aetivatable, translatable barnase expressioncassette (pTAB-BTR1) display atypical necrotic lesions at the site ofaphid feeding (most likely the result of barnase-induced cell death).These plants develop normally over the ensuing three months, incomparison to uninfected tobacco plants, and at no point developsymptoms characteristic of TYDV infection.

Total gDNA is isolated from leaves of each of the 20 transgenic tobaccoplants and uninfected controls, two weeks post-infection, using themethod of Stewart and Via (1993). Total gDNA (1 μg) is used as atemplate for a PCR with primers designed to the TYDV coat protein gene(CP-F and CP-R). The 765 by coat protein gene, and therefore virusgenome, is only detected in tobacco transformed with pTAB-BUTR1. Thisresult suggests that tobacco plants transformed with pTAB-BTR1 areresistant to TYDV infection and remain free of TYDV-induced symptomsover extended periods of time.

Primers:

CP-F 5′-ATGGCGGGCCGGTATAAGGGTTTGG-3′ [SEQ ID NO: 43] CP-R5′-TTATTGATTGCCAACTGATTTGAAAT-3′ [SEQ ID NO: 44]

Example 8 gfp Vector Constructions Based on BBTV

A similar series of vectors, based on the Rep-activatable barnasecassettes, were constructed using the reporter gene encoding greenfluorescent protein (GFP) and BBTV intergenic regions. Both expressionand re-circularization vectors were constructed by overlapping PCR in amanner similar to that of the pRTBN series of vectors and cloned into apUC19 vector. Primers used in the construction of these vectors areindicated in Table 5. In some cases plasmids pBN, pTBN6 and pTBN1 wereused as templates for PCRs.

TABLE 5 Oligonucleotide sequences Primer Name Sequence (5′-3′) +1 35SHAAGCTTCATGGAGTCAAAGA (SEQ ID NO: 45) +2 5′mGFPBamGGATCCATGAGTAAAGGAGAAGAACTT (SEQ ID NO: 46) +3 GFPB1AAGTCAAGTTTGAGGTAAGTTTCTGCTTC (SEQ ID NO: 47) −3 B2GFPGAAGCAGAAACTTACCTCAAACTTGACTT (SEQ ID NO: 448) +4 B1GFPTTGTTGATGTGCAGGGAGACACCCTCGTC (SEQ ID NO: 49) −4 GFPB2GACGAGGGTGTCTCCCTGCACATCAACAA (SEQ ID NO: 50) −5 3′mGFPSacGAGCTCTTATTTGTATAGTTCATCCAT (SEQ ID NO: 51) −6 NOS4-HAAGCTTTTCGCCATTCAGGCTGC (SEQ ID NO: 52) +7 B3-6TATCATTAATTAGTAAGTTGTGCTGTAA (SEQ ID NO: 53) −8 B4-6AATATTATATTACTCGCTTCTGCCTTCC (SEQ ID NO: 54) +9 B5-IATCATTAATTAGTCACACTATGACAAAAG (SEQ ID NO: 55) −10 B6-IAATATTATATTACTGATGCAAATGTCCCCG (SEQ ID NO: 56)

Initially, plasmids pGI (SEQ ID NO:69), pGI6 (SEQ ID NO:70), pGI1 (SEQID NO:71) were constructed and are shown in FIGS. 20, 21 and 22,respectively. Ultimately, plasmids pRGI6, pRGI1, pRGI1/6 wereconstructed and are shown in FIGS. 23, 24 and 25, respectively. In FIGS.20 to 25, intron refers to the potato ST-LS1 intron, intergenic regionsare derived from either BBTV DNA-1 or -6, and CT and NT refer to theC-terminal and N-terminal portions of the GFP reporter gene.

The efficacy of these various constructs is assessed in transient assaysvia micro-projectile bombardment of banana cell suspensions. The firsttransient assays, measure GFP expression to determine whether thevarious monomers are replicatively released and re-circularized, andthat the GFP gene is transcribed, processed and expressed correctly. ABBTV DNA-1 1.1 mer is mixed in equimolar amounts of either pGI1 or pGI6and bombarded into banana embryogenic cell suspensions. Eight dayspost-bombardment, replicative release and circularization are assayed bySouthern hybridization, using a GFP-specific probe. GFP expression ismonitored using a GFP microscope and quantified using Western blotanalysis and a GFP-specific antisera.

Southern hybridization indicates the presence of monomeric circularmolecules from either pGI1 or pGI6P only when the BBTV DNA-1 1.1mer isdelivered in trans. Similarly, GFP expression is only detected, when theviral-derived 1.1 mer is present.

Example 9 Stable Transformation of Banana for Barnase-Induced Resistanceto BBTV

Banana embryogenic cell suspensions of both Bluggoe and Cavendish areco-transformed with pRTBN6 and a plasmid carrying the selectable markergene using microprojectile bombardment (Becker et al., 2000).Regenerated plantlets are assayed by PCR to determine whether a completecopy of the Rep-activatable barnase cassette has been incorporated intothe banana genome. Positive transformants are multiplied and, initiallyfive plants from each transformation are challenged with 20 BBTVviruliferous aphids. Southern hybridization analyses are used to comparelevels of viral DNA accumulation between transformed and non-transformedplants. Promising transgenic lines are further multiplied, re-challengedand assayed.

In nearly all cases, transgenic banana plants show no evidence of bananabunchy top disease, in comparison to controls. Rather, atypical necrosisat the point of aphid feeding is observed, most likely reflectingbarnase-induced cell death. Using PCR and Southern hybridisation thecoat protein gene of BBTV can not be detected in any plant part tested,suggesting these lines are resistant to BBTV infection, replication andspread.

Example 10 Tissue-Specific and Inducible Rep-Activated Expression Basedon TYDV

In order to control the site of Rep expression, and therefore transgeneactivation/replication in planta, tissue-specific promoters wereemployed.

(a) Constitutive Expression

The vector pTAB16 contains a CaMV 35S pro-bar selection gene-ocs ter andCaMV 35S pro-uidA-CaMV 35S ter cassettes located between the right andleft T-DNA borders in pBIN16. The CaMV 35S-TYDV Rep gene cassette isexcised from p35S-Rep by EcoRI/BamHI digestion and inserted intosimilarly digested pTAB16 vector to replace the original CaMV 35S-uidAcassette. This construct is designated pTAB-TYDV-Rep.

(b) Seed-Specific Expression

The 1 kb rice glutelin promoter (Genbank Accession X52153) has beenshown to direct seed-specific reporter gene expression in tobacco (Leisyet al., 1989). The rice glutelin promoter is excised from the plasmidpGT3-JEFLK (Miller, 2001) by NcoI digestion and ligated into similarlydigested pTAB-TYDV-Rep vector to replace the original CaMV 35S promoter.This construct is designated pGL-TYDV-Rep.

(c) Root-Specific Expression

The 880 by Arabidopsis thaliana root-specific kinase homolog (ARSK1)promoter (Genbank Accession L22302) has been shown to directtissue-specific uidA reporter gene expression in epidermal,endoepidermal, and cortex regions of A. thaliana roots (Hwang andGoodman, 1995). The ARSK1 promoter is amplified from A. thaliana gDNA byPCR using primers ARSK-F and ARSK-R. The resulting PCR product is clonedinto pGEM-T and the sequence verified. The ARSK1 promoter is excisedfrom pGEM-T by NcoI digestion and ligated into similarly-digestedpTAB-TYDV-Rep to replace the original CaMV 35S promoter. This constructis designated pAR-TYDV-Rep.

Primers:

ARSK-F [SEQ ID NO: 57] 5′-CCATGGATCTCATTCTCCTTCAACAAGGCG-3′ ARSK-R[SEQ ID NO: 58] 5′-CCATGGTTTCAACTTCTTCTTTTGTGTTATTTG-3′

(d) Wound-Inducible Expression

The 2032 by Asparagus officinalis PR gene (AoPR1) promoter (GenbankAccession: A26573) has been shown to direct strong reporter geneexpression in wounded and actively dividing cell types such as, forexample, callus (Firek et al., 1993). The AoPR1 promoter is amplifiedfrom A. officinalis gDNA by PCR using primers AoPR-F and AoPR-R. Theresulting PCR product is cloned into pGEM-T and the sequence verified.The AoPR1 promoter is excised from pGEM-T by NcoI digestion and ligatedinto similarly digested pTAB-TYDV-Rep to replace the original CaMV 35Spromoter. This construct is designated pAo-TYDV-Rep.

Primers:

AoPR-F 5′-GAATTCAGGGGTAAGTTTGCAAATATC-3′ [SEQ ID NO: 59] AoPR-R5′-CGAGGTTGTGCCAGTCGAGCATTGCC-3′ [SEQ ID NO: 60]

(e) Alcohol-Inducible Expression

The ALC switch, derived from Aspergillus nidulans, is analcohol-inducible promoter system based on the AlcA promoter and AlcRreceptor. The ALC switch has been shown to function in plant systemsusing a uidA reporter gene model (Caddick et al., 1998). The plasmidpSRNAGS (BTI, Cornell University, Ithaca, N.Y.) contains a CaMV 358pro-AlcR gene-nos ter and AlcA pro-uidA reporter gene-CaMV 35S tercassette in pBIN16. The CaMV 358 pro-AlcR gene-nos ter-AlcA pro cassetteis amplified from pSRNAGS by PCR using primers 35S-IE (SEQ ID NO:21) andAlc-R (SEQ ID NO:61). The PCR product is cloned into pGEM-T and sequenceverified. The insert is then excised by NcoI digestion and ligated intosimilarly-digested pTAB-TYDV-Rep to replace the original CaMV 35Spromoter. This construct is designated pAlc-TYDV-Rep.

Primer:

Alc-R [SEQ ID NO: 61] 5′-CCATGGTTTGAGGCGAGGTGATAGGATTGG-3′

Each of the binary TYDV Rep-containing plasmids are introduced intoAgrobacterium tumefaciens (LBA4404) by electroporation using the methodof Singh et al. (1993).

(f) Tissue-Specific and Inducible Rep Expression Directs Reporter GeneActivation in Precise Tissue Types

Leaves from tobacco plants transformed with the plasmid pTEST4 aresuper-infected with Agrobacterium harbouring the plasmids pTAB-TYDV-Rep,pGL-TYDV-Rep, pAR-TYDV-Rep, pAo-TYDV-Rep, and pAlc-TYDV-Rep using themethod of Horsch et al. (1988). In this case, selection of transformedtobacco plants is achieved using the herbicide phosphoinothricinammonium (PPT). Transgenic plants are confirmed to contain the TYDV Repgene by PCR using the primers TYDV.RepF and TYDV.RepR. Ten independenttransformants for each plasmid are selected for further studies. Plantsare grown to maturity, allowed to flower and seed collected. Differentplant organs from independent transformants, including leaves, stems,roots, flower, and seed, are collected and GUS activity detected usinghistochemical assays.

Tobacco plants super-transformed with the plasmid pTAB-TYDV-Rep displaystrong GUS expression throughout all plant parts tested. The level ofGUS expression in these plants is considerably higher than plantstransformed with the non-replicating control, pTAB16.

In contrast, plants super-transformed with the plasmid pGL-TYDV-Rep showstrong GUS expression in the seeds only, plants super-transformed withthe plasmid pAR-TYDV-Rep show strong GUS expression in the roots only,plants super-transformed with the plasmid pAo-TYDV-Rep show strong GUSexpression in wounded and meristematic cells, and plantssuper-transformed with the plasmid pAlc-TYDV-Rep show strongconstitutive GUS expression when drenched in a 1% v/v ethanol solutiononly.

Rep-assisted nicking, joining and replication of the GUS expressioncassette is confirmed (as described previously) in all tissue types oftobacco plants super-transformed with the plasmid pTAB-TYDV-Rep. Incontrast, this activity is only detected in the seeds of plantssuper-transformed with the plasmid pGL-TYDV-Rep, in the roots of plantssuper-transformed with the plasmid pAR-TYDV-Rep, at the site of woundingin plants super-transformed with the plasmid pAo-TYDV-Rep, andconstitutively in ethanol-induced plants super-transformed with theplasmid pAlc-TYDV-Rep. This result suggests tissue-specific or inducedexpression of the TYDV Rep gene confers high-level expression from theRep-activatable GUS cassette in those tissue types only.

Example 11 MV p50 and hrmA Gene Mediated Resistance Based on TYDV (a)TYDV Rep-Activated Expression of the TMV p50 Helicase Fragment InducesSystemic Acquired Resistance (SAR) in Tobacco

Previous studies (Erickson et al., 1999) have demonstrated thatnon-viral expression of the 50 kDa tobacco mosaic virus (TMV) helicasefragment (p50) is sufficient to induce the N-mediated hypersensitiveresponse (HR) in suitable tobacco varieties (e.g. Nicotiana tabacum cv.“Petite Havana” SR1 homozygous for the N gene). The defense response ischaracterised by cell death at the site of virus infection and inductionof the systemic acquired resistance (SAR) pathway with resultinginhibition of viral replication and movement.

The p50 gene fragment is amplified by PCR using primers p50-F and p50-Rfrom cloned genomic TMV DNA (Plant Gene Expression Centre, University ofCalifornia, USA). The PCR product is cloned into pGEM-T and the sequenceverified. The catalase intron containing the TYDV LIR from pTEST3 (FIG.10) is engineered in frame into the unique EcoRI site in the p50 codingregion. This plasmid is designated pGEM50-LIR. A pUC-basedRep-activatable p50 gene plasmid is subsequently constructed, asdescribed for pTEST3 in Example 2, above. The cassette is inserted intopART27, as was described for pTEST4. The resulting p50 Rep-activatablebinary plasmid is designated pSAR1. The plasmid pSAR1 is used totransform Agrobacterium as previously described.

Primers:

p50-F [SEQ ID NO: 62] 5′-CCATGGAGATAGAGTCTTTAGAGCAGTTTC-3′ p50-R[SEQ ID NO: 63] 5'-GGATCCTATTGTGTTCCTGCATCGACCTTA-3'

(b) Systemic Acquired Resistance to TYDV in Tobacco

Ten tobacco plants (Nicotiana tabacum cv. “Petite Havana” SR1 homozygousfor the N gene) transformed with T-DNA from plasmid pSAR1 are obtainedby Agrobacterium-mediated transformation and confirmed to contain theRep-activatable cassette and NPTII gene by PCR as described above.Plants are infected with TYDV and observed for symptoms, as previouslydescribed. Tobacco plants transformed with the Rep-activatable, p50 genedisplay atypical hypersensitive necrosis at the site of aphid feeding,two days post infection. These plants develop normally over the ensuing3 months, in comparison to infected non-transgenic tobacco plants, whichdisplay typical TYDV-induced symptoms.

TYDV genomic DNA is detected in inoculated non-transgenic tobacco butnot in transgenic nor uninfected control lines, as previously described.This result suggests TYDV Rep-induced expression of the TMV p50 gene issufficient to stimulate the N gene hypersensitive response in suitabletobacco cultivars and provide resistance to TYDV infection.

(c) Wound-Inducible TYDV Rep Expression Activates TMV p50 GeneExpression and Triggers SAR to a Variety of Pathogens.

Leaves from pSAR1-transformed tobacco plants are super-infected withAgrobacterium containing the plasmid pAo-TYDV-Rep (Example 10), aspreviously described. Ten super-transformed lines, confirmed to containthe wound-inducible Rep gene, are selected for further studies.Transgenic plants and suitable controls are subjected to infection witha variety of viral pathogens e.g. (1) aphid transmission of tobacco veinmottling virus, (ii) tobacco rattle virus via the nematode vectorParatrichodorus pachydermus, and (iii) biolistic introduction of aninfectious BeYDV 1.1mer. Over time, plants are observed forcharacteristic viral-induced symptoms. All transgenic plants displayatypical hypersensitive necrosis at the site of virus inoculation incomparison to controls.

(d) Wound-Inducible Expression of TYDV Rep Activates hrmA-Mediated BroadRange Pathogen Resistance in Tobacco

The hrmA gene product from Pseudomonas syringae pv. syringae has beenshown to activate pathogen related genes in a number of tobaccocultivars, and confer resistance to variety of pathogens, includingviruses, fungi and bacteria (Shen et al., 2000).

The hrmA gene is amplified from a plasmid containing the coding sequenceby PCR using primers hrm-F and hrm-R. A pUC-based Rep-activatable hrmAplasmid is subsequently constructed, as described in Example 2, above,for pTEST3. The cassette is inserted into pART27, as described forpTEST4. The resulting hrmA-activatable binary plasmid is designatedpSAR2. The plasmid pSAR2 is used to transform Agrobacterium, asdescribed.

hrm-F [SEQ ID NO: 64] 5′-CCATGGGCATGCACGCTTCTCCAGCGTAGAAGCG-3′ hrm-R[SEQ ID NO: 65] 5′-GGATCCTCAGTTTCGCGCCCTGAGCGCCGG-3′

Ten tobacco plants (Nicotiana tabacum) transformed with T-DNA fromplasmid pSAR2 are obtained by Agrobacterium-mediated transformation andconfirmed to contain the Rep-activatable cassette and NPTII gene by PCRas previously described. Leaves from pSAR2 transformed tobacco plantsare super-infected with Agrobacterium containing the plasmidpAo-TYDV-Rep, as previously described. Ten super-transformed lines,confirmed to contain the wound-inducible Rep gene, are selected forfurther studies. Transgenic plants and suitable controls are subjectedto infection with a variety of pathogens e.g. tobacco vein mottlingvirus, tobacco etch virus, blank shank fungus Phytophthora parasitica,and wild fire bacterium Pseudomonas syrinagae pv. tabaci. Over time,plants are observed for characteristic pathogen-induced symptoms. Alltransgenic plants display atypical hypersensitive necrosis at the siteof inoculation, in comparison to controls.

Example 12 Over Expression of Human Serum Albumin Based on TYDV (a)Rep-Activated, Over-Expression of a Commercially Important Protein,Human Serum Albumin

Albumin is a soluble, monomeric protein which comprises about one-halfof the blood serum protein. Albumin functions primarily as a carrierprotein for steroids, fatty acids, and thyroid hormones and plays a rolein stabilizing extracellular fluid volume.

The 1831 by Human serum albumin (HSA) coding region (Lawn et al., 1981)is amplified from a plasmid containing the gene by PCR using primersAlb-F and Alb-R. A pUC-based Rep-activatable HSA plasmid is subsequentlyconstructed, as described in Example 2, above, for pTEST3. The cassetteis inserted into pART27, as described for pTEST4. The resultingHSA-activatable binary plasmid is designated pHSA1. The plasmid pHSA1 isused to transform Agrobacterium, as described.

Ten tobacco plants (Nicotiana tabacum) transformed with T-DNA fromplasmid pHSA1 are obtained by Agrobacterium-mediated transformation andconfirmed to contain the Rep-activatable cassette and NPTII gene by PCR,as described. Leaves from pHSA1 transformed tobacco plants aresuper-infected with Agrobacterium containing plasmids pTAB-TYDV-Rep,pGL-TYDV-Rep, pAo-TYDV-Rep, pAlc-TYDV-Rep, as described. Tensuper-transformed lines from each transformation are confirmed tocontain the Rep gene, and selected for further studies. Plants are grownto maturity and HSA content assessed using ELISA and antibodies raisedto the HSA gene product. HSA protein, is detected at high levels but indifferent plant parts or under specific conditions; i.e. constitutively(pTAB-TYDV-Rep), seed only (pGL-TYDV-Rep), wounded tissues(pAo-TYDV-Rep) and constitutively in alcohol treated plants(pAlc-TYDV-Rep). A proposed model for Rep-activated expression of thehuman serum albumin gene from the plasmid pHSA1 is depicted in FIG. 26.

Primers:

Primers: Alb-F [SEQ ID NO: 72] 5′-CCATGGAGATGAAGTGGGTAACCTTTATTTCC-3′Alb-R [SEQ ID NO: 73] 5′-GGATCCTTATAAGCCTAAGGCAGCTTGACT-3′

Those skilled in, the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

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1. A construct comprising a genetic element operably flanked bynucleotide sequences recognizable by a viral-derived,replication-facilitating protein or its derivatives or eukaryotic orprokaryotic cell homologues when integrated into the genome of aeukaryotic cell which viral-derived, replication-facilitating protein orits derivatives or eukaryotic or prokaryotic cell homologues facilitatesexcision and circularization of the genetic element and all or part ofthe flanking nucleotide sequences and wherein said nucleotide sequencesrecognizable by said viral-derived, replication-facilitating protein orits derivatives or eukaryotic or prokaryotic cell homologues areadjacent to or inserted within one or more extraneous sequencesincluding intron sequences or parts thereof or other splice signalswherein the genetic element and other nucleotide sequences, in anon-circular form, comprise two modular nucleotide sequences which, uponcircularization, form a genetic sequence exhibiting a property or acapacity for exhibiting a property absent in the two modular nucleotidesequences prior to circularization or prior to circularization andexpression.
 2. The construct of claim 1 wherein the viral-derivedreplicating protein is Rep or a derivative or functional equivalent orhomologue thereof.
 3. The construct of claim 2 wherein the viral-derivedreplicating protein is Rep.
 4. The construct of claim 1 wherein theviral-derived replication-facilitating protein and the flankingnucleotide sequences are from the same virus.
 5. The construct of claim1 wherein the viral-derived replication-facilitating protein and theflanking nucleotide sequences are from different virus.
 6. The constructof claim 1 wherein the extraneous sequences comprise a coding sequence,a promoter and one or more splice signals.
 7. The construct of claim 6wherein the coding sequences, when the construct is in linear form,comprises two modular sequences separated by an intronic sequence. 8.The construct of claim 6 wherein, after circularization of the geneticelement, a full coding sequence is operably linked to said promoter. 9.The construct of claim 8 wherein the coding sequence encodes an mRNA orpeptide, polypeptide or protein which confers a phenotypic or genotypicproperty on said call or on an organism or plant comprising said cell.10. The construct of claim 9 wherein the peptide, polypeptide or proteincauses or otherwise facilitates cell death.
 11. The construct of claim 1wherein the construct is introduced to a eukaryotic cell.
 12. Theconstruct of claim 11 wherein the eukaryotic cell is a plant cell. 13.The construct of claim 11 wherein the eukaryotic call is a mammaliancell.
 14. The construct of claim 11 wherein the eukaryotic cell is anavian cell.
 15. A construct comprising a genetic element flanked byRep-protein recognition sequences or functional homologues from otherviruses or eukaryotic or prokaryotic cells which facilitate thegeneration of a circular nucleotide sequence comprising said geneticelement in the presence of a Rep protein or its functional derivativesor homologues wherein said Rep-protein recognition sequences areadjacent to or inserted within one or more splice signals, said geneticelement comprising a polynucleotide sequence operably linked toregulatory sequences required to permit expression of saidpolynucleotide sequence when said genetic element is contained within acircularized molecule wherein the genetic element in linear formcomprises in the 5′ to 3′ order:— A polynucleotide sequence; andregulatory sequences to permit expression of said polynucleotidesequence when in circular form; such that upon circularization thegenetic element comprises the regulatory sequence separated from thepolynucleotide sequence by all or part of a Rep protein-recognitionsequence wherein upon expression, said polynucleotide sequence encodesan expression product.
 16. The construct of claim 15 wherein thepolynucleotide sequences, when the construct is in linear form,comprises 5′ and 3′ portions of a coding sequence separated by a splicesignal such that in circular form, a full coding sequence isconstituted.
 17. The constrict of claim 15 wherein a single Repprotein-recognition sequence is employed.
 18. The construct of claim 15wherein two Rep protein-recognition sequences derived from differentviruses are employed.
 19. The construct of claim 15 wherein thepolynucleotide sequence further comprises a terminator sequence operablylinked to the 3′ end of the coding sequence.
 20. The construct of claim15 wherein the regulatory sequence is a promoter operably linked to the5′ end of the coding sequence.
 21. The construct of claim 20 wherein thecoding sequence, when the genetic element is in circular form, encodesan mRNA or a peptide, polypeptide or protein which confers a phenotypicor genotypic property on said cell or on an organism or plant comprisingsaid cell.
 22. The construct of claim 21 wherein the peptide,polypeptide or protein causes or otherwise facilitates cell death. 23.The construct of claim 15 wherein the construct is introduced to aeukaryotic cell.
 24. The construct of claim 23 wherein the eukaryoticcell is a plant cell.
 25. The construct of claim 23 wherein theeukaryotic cell is a mammalian cell.
 26. The construct of claim 23wherein the eukaryotic cell is an avian cell.
 27. The constructcomprising in 5′ to 3′ order first, second, third, fourth, fifth andsixth nucleotide sequences wherein: the first and sixth nucleotidesequences may be the same or different and each comprises a Repprotein-recognition sequence capable of being recognized by one or moreRep proteins or derivatives or homologues thereof such that geneticmaterial flanked by said first and sixth sequences including all or partof said first and sixth sequences when said construct is integrated in alarger nucleotide sequence is capable of being excised and circularizedwherein said Rep-protein recognition sequences are adjacent to orinserted within one or more extraneous sequences including intronicsequences or parts thereof or other splice signals; the secondnucleotide sequence comprises a 3′ portion of a polynucleotide sequence;the third nucleotide sequence is a transcription terminator orfunctional derivative or homologue thereof operably linked to saidsecond sequence; the fourth nucleotide sequence is a promoter sequenceoperably linked to the fifth nucleotide sequence; and the fifthnucleotide sequence is a 5′ portion of a polynucleotide sequence whereinthe 5′ and 3′ portions of said polynucleotide sequence represent a fullcoding sequence of said polynucleotide sequence; wherein in the presenceof one or more Rep proteins, when the construct is integrated into alarger nucleotide sequence, a circularized genetic sequence is generatedseparate from said larger nucleotide sequence comprising in order saidpromoter sequence operably linked to a polynucleotide sequencecomprising all or part of the extraneous sequence or other splice signalcomprising all or part of one or more of said first and sixth nucleotidesequences and a transcription terminator sequence.
 28. The construct ofclaim 27 wherein the full coding sequence of the polynucleotide sequenceencodes an mRNA or a peptide, polypeptide or protein which confers aphenotypic or genotypic property on said cell or on an organism or plantcomprising said cell.
 29. The construct of claim 28 wherein the peptide,polypeptide or protein causes or otherwise facilitates cell death. 30.The construct of claim 27 wherein the construct is introduced into aeukaryotic cell.
 31. The construct of claim 30 wherein the eukaryoticcell is a plant cell.
 32. The construct of claim 30 wherein theeukaryotic cell is a mammalian cell.
 33. The construct of claim 30wherein the eukaryotic cell is an avian cell.
 34. A method forgenerating a transgenic plant or progeny thereof resistant to a ssDNAvirus, said method comprising introducing into the genome of said planta construct comprising in the 5′ to 3′ order, a Rep protein-recognitionsequence adjacent to or within an intronic sequence or other splicesignal, a 3′ end portion of a polynucleotide sequence, a transcriptionterminator or its functional equivalent, a promoter sequence operablylinked to a 5′ end portion of the polynucleotide sequence wherein the 5′and 3′ portions of the polynucleotide sequence represent the codingregion of a peptide, polypeptide or protein capable of inducing celldeath or dormancy, and same or different Rep protein-recognitionsequences; wherein upon infection of said plant cells by a ssDNA virushaving a Rep protein which is capable of recognizing the flanking Repprotein-recognition sequences, the construct is excised and circularizesthus reconstituting said polynucleotide sequence in a form which isexpressed into a peptide, polypeptide or protein which kills the plantcell or otherwise renders the plant cell dormant.
 35. The method ofclaim 34 wherein the ssDNA virus is a memberof the Geminiviridae ornanovirus group.
 36. The method according to claim 35 wherein theGeminiviridae virus is a begomovirus or mastrevirus.
 37. The method ofclaim 34 wherein the construct comprises the nucleotide sequencesubstantially as set forth in SEQ ID NO:31 to SEQ ID NO:36 or anucleotide sequence having 60% similarity to each of SEQ ID NO:31 to SEQID NO:36 or a nucleotide sequence capable of hybridizing to one or moreof SEQ ID NO:31 to SEQ ID NO:36 or a complementary form thereof underlow stringency conditions at 42° C.
 38. A linear genetic element for usein generating a covalently closed circular DNA construct, said geneticelement comprising a 5′ to 3′ direction, a 3′ portion of apolynucleotide sequence operably linked to a transcription terminator; apromoter operably linked to a 5′ portion of a polynucleotide sequencewherein upon circularization, the 5′ portion of the polynucleotidesequence is operably linked to said 3′ portion of the polynucleotidesequence separated by all or part of an extraneous sequence or intronsequence or other splice signal.
 39. The genetic element of claim 38further comprising nucleotide sequences recognizable by a viral-derived,replication-facilitating protein or its derivatives or eukaryotic orprokaryotic homologues.
 40. The genetic element of claim 39 wherein theviral-derived, replication-facilitating protein is Rep or a derivativeor functional equivalent or homologue thereof.
 41. The genetic elementof claim 40 wherein the viral-derived, replication-facilitating proteinis Rep.
 42. The genetic element of claim 39 wherein two nucleotidesequences recognized by viral-derived, replication-facilitating proteinsand derived from different viruses are employed.
 43. The genetic elementof claim 38 wherein the polynucleotide sequence, when in the covalentlyclosed, circular construct encodes an mRNA or a peptide, polypeptide orprotein.
 44. The genetic element of claim 42 wherein the peptide,polypeptide or protein causes or otherwise facilitates cell death. 45.The genetic element of claim 38 when introduced into a eukaryotic cell.46. The genetic element of claim 44 wherein the eukaryotic cell is aplant cell.
 47. The genetic element of claim 44 wherein the eukaryoticcell is a mammalian cell.
 48. The genetic element of claim 44 whereinthe eukaryotic cell is an avian cell.
 49. A genetically modified plantor part thereof comprising a genetic construct of claim
 1. 50. Agenetically modified plant or part thereof comprising a geneticconstruct of claim
 15. 51. A genetically modified plant or part thereofcomprising a genetic construct of claim
 27. 52. A genetically modifiedplant or part thereof comprising a genetic element of claim
 38. 53. Agenetically modified avian species comprising a genetic construct ofclaim
 1. 54. A genetically modified avian species comprising a geneticconstruct of claim
 15. 55. A genetically modified avian speciescomprising a genetic construct of claim
 27. 56. A genetically modifiedplant or part thereof comprising a genetic element of claim
 38. 57. Aconstruct comprising a genetic element flanked by Repprotein-recognition sequences which facilitate the generation of acircular nucleotide sequence comprising said genetic element in thepresence of a Rep protein or its functional derivatives or homologueswherein said Rep-protein recognition sequences are adjacent to orinserted within one or more extraneous sequences including intronicsequences or parts thereof or other splice signal, said genetic elementcomprising a 3′ portion and a 5′ portion of a promoter separated by alength of a nucleotide sequence to substantially prevent functioning ofsaid promoter, said genetic element in linear form comprises in the 5′to 3′ order:— a 3′ portion of said promoter; optionally a polynucleotidesequence operably linked to said 3′ portion of said promoter; and a 5′portion of said promoter, said that upon circularization the geneticelement comprises the 5′ and 3′ portions of the promoter sequenceseparated by all or part of one or more of a Rep protein-recognitionsequence, an intron sequence or other splice signal that does notinactivate the activity of the promoter, said circular moleculeoptionally further comprising the promoter operably linked to apolynucleotide sequence.